Born in Charles County, Maryland, Mudd was the fourth of ten children of Henry Lowe and Sarah Ann Reeves Mudd. He grew up on "Oak Hill", his father's tobacco plantation of several hundred acres which was located 30 miles (48 km) southeast of downtown Washington, D.C., and which was worked by 89 slaves.

At the age of 15, after several years of home tutoring, Mudd went off to boarding school at St. Johns in Frederick, Maryland. Two years later, he enrolled at Georgetown College in Washington, D.C.. He then studied medicine at the University of Maryland, Baltimore, writing his thesis on dysentery.

Upon graduation in 1856, Mudd returned to Charles County to practice medicine, marrying his childhood sweetheart, Sarah Frances (Frankie) Dyer Mudd one year later. As a wedding present, Mudd's father gave the couple 218 acres (0.88 km²) of his best farmland, known as St. Catherine’s, and a new house. While the house was under construction, the young Mudds lived with Frankie's bachelor brother, Jeremiah Dyer, finally moving into their new home in 1859. They had nine children in all; four before Mudd's arrest and five after his release from prison. To supplement his income from his medical practice, Mudd became a small scale tobacco grower, using five slaves according to the 1860 U.S. Slave Census. Mudd believed that slavery was divinely ordained, writing a letter to the theologian Orestes Brownson to that effect.

With the advent of the American Civil War in 1861, the Southern Maryland slave system and the economy it supported rapidly began to collapse. In 1863, the Union Army established Camp Stanton just 10 miles (16 km) from the Mudd farm to enlist black freemen and run away slaves. Six regiments totaling over 8,700 black soldiers, many from Southern Maryland, were trained there. In 1864, Maryland, which was exempt from Lincoln's 1863 Emancipation Proclamation, abolished slavery, making it difficult for growers like Mudd to operate their plantations. As a result, Mudd considered selling his farm and depending on his medical practice. As Mudd pondered his alternatives, he was introduced to someone who said he might be interested in buying his property, a 26 year old actor by the name of John Wilkes Booth.

Many historians agree that President Abraham Lincoln's future assassin, John Wilkes Booth, visited Bryantown, Maryland, in November and December 1864, claiming to look for real estate investments. Bryantown is about 25 miles (40 km) from Washington, D.C., and about 5 miles (8.0 km) from Mudd’s farm. The real estate story was merely a cover; Booth’s true purpose was to plan an escape route as part of a plan to kidnap Lincoln. Booth believed the federal government would ransom Lincoln by releasing a large number of Confederate prisoners of war.

Historians do agree that Booth met Mudd at St. Mary’s Catholic Church in Bryantown during one of those visits, probably the November visit. Booth visited Mudd at his farm the next day, and stayed there overnight. The following day, Booth purchased a horse from Mudd’s neighbor and returned to Washington. Some historians believe that Booth used his visit to Bryantown to recruit Mudd to his kidnapping plot, although others believe that Mudd would have had no interest in such a scheme.

A short time later, on December 23, 1864, Mudd went to Washington where he met Booth again. Some historians believe the meeting was pre-arranged; others believe it was accidental. The two men, plus John Surratt, Jr. and Louis J. Weichmann, had a conversation and drinks together, first at Booth’s hotel, and later at Mudd’s.

According to a statement made by co-conspirator George Atzerodt, found long after his death and taken down while he was in federal custody on May 1, 1865, Mudd knew in advance about Booth's plans; Atzerodt was sure the doctor knew, he said, because Booth had "sent (as he told me) liquors & provisions [...] about two weeks before the murder to Dr. Mudd's".

After Booth shot President Lincoln on April 14, 1865, he broke his left leg while fleeing Ford's Theater. Booth met up with David Herold and together they made for Virginia via Southern Maryland. They stopped at Mudd's house around four o'clock in the morning on April 15. Mudd set, splinted and bandaged Booth's broken leg, and arranged for a carpenter, John Best, to make a pair of crutches for Booth. "I had no proper paste - board for making splints... so..I... took a piece of bandbox and split it in half, doubled it at right angles, and took some paste and pasted it into a splint". Booth and Herold spent between twelve and fifteen hours at Mudd's house. They slept in the front bedroom on the second floor. It is unclear whether Mudd had been informed that Booth had murdered President Lincoln at that point.

Mudd went to Bryantown during the day on April 15 to run errands; if he did not already know the news of the assassination from Booth, he certainly learned of it on this trip. He returned home that evening, and accounts differ as to whether Booth and Herold had already left, whether Mudd met them as they were leaving, or whether they left at Mudd's urging and with his assistance.

Whichever is true, Mudd did not immediately contact the authorities. When questioned, he stated that he had not wanted to leave his family alone in the house lest the assassins return and find him absent and his family unprotected. He waited until Mass the following day, Easter Sunday, when he asked his second cousin, Dr. George Mudd — a resident of Bryantown — to notify the 13th New York Cavalry in Bryantown under the command of Lieutenant David Dana. This delay in contacting the authorities drew suspicion and was a significant factor in tying Mudd to the conspiracy.

During his initial investigative interview on April 18, Mudd stated that he had never seen either of the parties before. In his sworn statement of April 22, he told about Booth's visit to Bryantown in November 1864, but then said "I have never seen Booth since that time to my knowledge until last Saturday morning." He deliberately hid the fact of his meeting with Booth in Washington in December 1864. In prison, Mudd belatedly admitted the Washington meeting, saying he ran into Booth by chance during a Christmas shopping trip. Mudd’s failure to mention the meeting in his sworn statement to detectives was a big mistake. When Louis J. Weichmann later told the authorities of this meeting, they realized Mudd had misled them, and immediately began to treat him as a suspect rather than a witness. During the conspiracy trial, Lieutenant Alexander Lovett testified that "On Friday, the 21st of April, I went to Mudd's again, for the purpose of arresting him. When he found we were going to search the house, he said something to his wife, and she went up stairs and brought down a boot. Mudd said he had cut it off the man's leg. I turned down the top of the boot, and saw the name 'J. Wilkes' written in it."

After Booth's death (April 26, 1865), Mudd was arrested and charged with conspiracy to murder Abraham Lincoln.

On May 1, 1865, President Andrew Johnson ordered the formation of a nine man military commission to try the conspirators. Mudd was represented by General Thomas Ewing, Jr.. The trial began on May 10, 1865. Mary Surratt, Lewis Powell, George Atzerodt, David Herold, Samuel Mudd, Michael O'Laughlen, Edmund Spangler and Samuel Arnold were all charged with conspiring to murder Lincoln. The prosecution called 366 witnesses.

The defense sought to prove that Mudd was a loyal citizen, citing his self - description as a "Union man" and asserting that he was "a deeply religious man, devoted to family, and a kind master to his slaves". The prosecution presented witnesses who testified that he had shot one of his slaves in the leg, and threatened to send others to Richmond, Virginia, to assist in the construction of Confederate defenses. The prosecution also contended that he had been a member of a Confederate communications distribution agency and had sheltered Confederate soldiers on his plantation.

On June 29, 1865, Mudd was found guilty with the others. The testimony of Louis J. Weichmann was crucial in obtaining the convictions. According to the historian Edward Steers, the testimony presented by former slaves was also crucial, although it faded from public memory. Mudd escaped the death penalty by one vote and was sentenced to life imprisonment. Four of the defendants, Surratt, Powell, Atzerodt and Herold, were hanged at the Old Penitentiary at the Washington Arsenal on July 7, 1865.

Mudd, O'Laughlen, Arnold and Spangler were imprisoned at Fort Jefferson in the Dry Tortugas about 70 miles (110 km) west of Key West, Florida. The fort housed Union Army deserters and held about six hundred prisoners when Mudd and the others arrived. Prisoners lived on the second tier of the fort, in unfinished open air gun rooms called casemates. Mudd and his three companions lived in the casemate directly above the fort's main entrance, called the Sally Port.

In September 1865, two months after Mudd arrived, control of Fort Jefferson was transferred from the 161st New York Volunteers to the 82nd United States Colored Infantry. On September 25, 1865, he attempted to escape from Fort Jefferson by stowing away on the transport Thomas A. Scott. Some historians believe that as a recent slave owner and a person convicted of conspiring to kill the president whose presidency led to the freeing of the slaves, Mudd was fearful of his treatment by the incoming 82nd United States Colored Infantry and this is why he made his bid for freedom. Others, including Edward Steers Jr., believe it was because he remained prejudiced against blacks and felt humiliated being guarded by them. He was quickly discovered and placed in the fort's guardhouse. On October 18, he was transferred along with Samuel Arnold, Michael O’Laughlen, Edman Spangler, and George St. Leger Grenfell to a large empty ground level gunroom the soldiers referred to as "the dungeon". Mudd and the others were let out of the dungeon six days a week to work around the fort. On Sundays and holidays they were confined inside. The men wore leg irons while working outside, but the irons were removed when inside the dungeon.

After three months in the dungeon, Mudd and the others were returned to the general prison population. However, because of his attempted escape, Mudd lost his privilege of working in the prison hospital and was assigned to work in the prison carpentry shop with Spangler.

There was an outbreak of yellow fever in the fall of 1867 at the fort. Michael O'Laughlen eventually died of it on September 23. The prison doctor died and Mudd agreed to take over the position. In this role he was able to help stem the spread of the disease. The soldiers in the fort wrote a petition to President Johnson in October 1867 stating of Mudd's assistance, "He inspired the hopeless with courage and by his constant presence in the midst of danger and infection... doubtless owe their lives to the care and treatment they received at his hands." Probably as a reward for his work in the yellow fever epidemic, Dr. Mudd was reassigned from the carpentry shop to a clerical job in the Provost Marshall's office, where he remained until his pardon.

Due in part to the influence of his defense attorney, Thomas Ewing Jr., who was influential in the President's administration, on February 8, 1869, Mudd was pardoned by President Andrew Johnson. He was released from prison on March 8, 1869 and returned home to Maryland on March 20, 1869. On March 1, 1869, three weeks after he pardoned Mudd, Johnson also pardoned Spangler and Arnold.

When Mudd returned home, well wishing friends and strangers, as well as inquiring newspaper reporters, besieged him. Mudd was very reluctant to talk to the press because he felt they had misquoted him in the past. He gave one interview after his release to the New York Herald, but immediately regretted it, complaining that the article had several factual errors and that it misrepresented his work during the yellow fever epidemic. On the whole though, Mudd continued to enjoy the friendship of his friends and neighbors. He resumed his medical practice and slowly brought the family farm back to productivity.

In 1873, Spangler traveled to the Mudd farm, where Mudd and his wife welcomed him as the friend whom Mudd credited with saving his life while suffering with yellow fever at Fort Jefferson. Spangler lived with the Mudd family for about eighteen months, earning his keep by doing carpentry, gardening, and other farm chores, until his death on February 7, 1875.

Mudd always had an interest in politics. While in prison, he stayed abreast of political happenings through the newspapers he was sent. After his release, he became active again in community affairs. In 1874, he was elected chief officer of the local farmers association, the Bryantown Grange. In 1876, he was elected Vice President of the local Democratic Tilden - Hendricks presidential election committee. Tilden lost that year to Republican Rutherford B. Hayes in a hotly disputed election. The next year Mudd ran as a Democratic candidate for the Maryland House of Delegates, but was defeated by the popular Republican William Mitchell.

Mudd’s ninth child, Mary Eleanor “Nettie” Mudd, was born in 1878. That same year, he and his wife temporarily took in a seven year old orphan named John Burke, one of 300 abandoned children sent to Maryland families from the New York City Foundling Asylum run by the Catholic Sisters of Charity. The Burke boy was permanently settled with farmer Ben Jenkins.

In 1880, the Port Tobacco Times reported that Mudd’s barn containing almost eight thousand pounds of tobacco, two horses, a wagon, and farm implements was destroyed by fire.

Mudd was just 49 years old when he died of pneumonia on January 10, 1883. He is buried in the cemetery at St. Mary’s Catholic Church in Bryantown, the same church where he once met up with John Wilkes Booth.

Mudd's grandson, Dr. Richard Mudd, tried unsuccessfully to clear his grandfather's name from the stigma of aiding John Wilkes Booth. In 1951, he published The Mudd Family of the United States, an encyclopedic two volume history of the Mudd family in America, beginning with Thomas Mudd who arrived from England in 1665. A second edition was published in 1969.

Richard Mudd petitioned several successive Presidents, receiving replies from Presidents Jimmy Carter and Ronald Reagan. Carter, while sympathetic, responded that he had no authority under law to set aside the conviction; Reagan that he had come to believe that Samuel Mudd was innocent of any wrongdoing. In 1992 Representatives Steny Hoyer and Thomas W. Ewing introduced House Bill 1885 to overturn the conviction, but it failed while in committee. Mudd then turned to the Army Board for Correction of Military Records, which recommended that the conviction be overturned on the basis that Mudd should have been tried by a civilian court. The recommendation was rejected by Acting Assistant Secretary of the Army William D. Clark. Several other legal venues were attempted, ending in 2003 when the U.S. Supreme Court refused the case, stating that the deadline for filing had been missed.

St. Catharine, also known as the Dr. Samuel A. Mudd House, was listed on the National Register of Historic Places in 1974.

Mudd's life was the subject of a 1936 John Ford directed film The Prisoner of Shark Island, based on a script by Nunnally Johnson. Another film, entitled The Ordeal of Dr. Mudd, was made in 1980. It starred Dennis Weaver as Mudd, and espoused the point of view that Mudd was innocent of any conspiracy.

Roger Mudd, an Emmy Award winning journalist and television host, is related to Samuel Mudd, though he is not a direct descendant, as has been mistakenly reported.

On the episode Swiss Diplomacy of the NBC Drama The West Wing, first lady (and cardiac surgeon) Dr. Abby Bartlet commented on the duty of a physician to treat an injured patient despite potential legal repercussions. She responded to Mudd's treason conviction with "So that's the way it goes. You set the leg".

Samuel Mudd is sometimes given as the origin of the phrase "your name is mud", as in, for example, the 2007 film National Treasure: Book of Secrets. However, according to an online etymology dictionary, this phrase has its earliest known recorded instance in 1823, ten years before Mudd's birth, and is based on an obsolete sense of the word "mud" meaning "a stupid twaddling fellow".