In Pasteur's early work as a chemist, he resolved a problem concerning the nature of tartaric acid (1848). A solution of this compound derived from living things (specifically, wine lees) rotated the plane of polarization of light passing through it. The mystery was that tartaric acid derived by chemical synthesis had no such effect, even though its chemical reactions were identical and its elemental composition was the same. This was the first time anyone had demonstrated chiral molecules.

Pasteur's doctoral thesis on crystallography attracted the attention of W.T. Fuillet and he helped Pasteur garner a position of professor of chemistry at the Faculté (College) of Strasbourg.

In the year of 1854, Louis was named Dean of the new Faculty of Sciences in Lille. (It was on this occasion that Pasteur uttered his oft-quoted remark: "...dans les champs de l'observation, le hasard ne favorise que les esprits préparés" (In the field of observation, chance favors only the prepared mind.)) In 1856, he was made administrator and director of scientific studies of the École Normale Supérieure.

Pasteur demonstrated that fermentation is caused by the growth of micro-organisms, and that the emergent growth of bacteria in nutrient broths is not due to spontaneous generation but rather to biogenesis (Omne vivum ex ovo). He exposed boiled broths to air in vessels that contained a filter to prevent all particles from passing through to the growth medium, and even in vessels with no filter at all, with air being admitted via a long tortuous tube that would not allow dust particles to pass. Nothing grew in the broths unless the flasks were broken open; therefore, the living organisms that grew in such broths came from outside, as spores on dust, rather than spontaneously generated within the broth. This was one of the last and most important experiments disproving the theory of spontaneous generation. The experiment also supported germ theory.

While Pasteur was not the first to propose germ theory (Girolamo Fracastoro, Agostino Bassi, Friedrich Henle and others had suggested it earlier), he developed it and conducted experiments that clearly indicated its correctness and managed to convince most of Europe it was true. Today he is often regarded as the father of germ theory and bacteriology, together with Robert Koch.

Pasteur's research also showed that the growth of micro-organisms was responsible for spoiling beverages, such as beer, wine and milk. With this established, he invented a process in which liquids such as milk were heated to kill most bacteria and moulds already present within them. He and Claude Bernard completed the first test on April 20, 1862. This process was soon afterwards known as pasteurization.

Beverage contamination led Pasteur to the idea that micro-organisms infecting animals and humans cause disease. He proposed preventing the entry of micro-organisms into the human body, leading Joseph Lister to develop antiseptic methods in surgery.

In 1865, two parasitic diseases called pébrine and flacherie were killing great numbers of silkworms at Alais (now Alès). Pasteur worked several years proving it was a microbe attacking silkworm eggs which caused the disease, and that eliminating this microbe within silkworm nurseries would eradicate the disease.

Pasteur also discovered anaerobiosis, whereby some micro organisms can develop and live without air or oxygen, called the Pasteur effect.

Pasteur's later work on diseases included work on chicken cholera. During this work, a culture of the responsible bacteria had spoiled and failed to induce the disease in some chickens he was infecting with the disease. Upon reusing these healthy chickens, Pasteur discovered that he could not infect them, even with fresh bacteria; the weakened bacteria had caused the chickens to become immune to the disease, even though they had only caused mild symptoms.

His assistant Charles Chamberland (of French origin) had been instructed to inoculate the chickens after Pasteur went on holiday. Chamberland failed to do this, but instead went on holiday himself. On his return, the month old cultures made the chickens unwell, but instead of the infection being fatal, as it usually was, the chickens recovered completely. Chamberland assumed an error had been made, and wanted to discard the apparently faulty culture when Pasteur stopped him. Pasteur guessed the recovered animals now might be immune to the disease, as were the animals at Eure-et-Loir that had recovered from anthrax.

In the 1870s, he applied this immunization method to anthrax, which affected cattle, and aroused interest in combating other diseases. Pasteur publicly claimed he had made the anthrax vaccine by exposing the bacillus to oxygen. His laboratory notebooks, now in the Bibliothèque Nationale in Paris, in fact show Pasteur used the method of rival Jean-Joseph-Henri Toussaint, a Toulouse veterinary surgeon, to create the anthrax vaccine. This method used the oxidizing agent potassium dichromate. Pasteur's oxygen method did eventually produce a vaccine but only after he had been awarded a patent on the production of an anthrax vaccine.

The notion of a weak form of a disease causing immunity to the virulent version was not new; this had been known for a long time for smallpox. Inoculation with smallpox was known to result in far less scarring, and greatly reduced mortality, in comparison with the naturally acquired disease. Edward Jenner had also discovered vaccination, using cowpox to give cross immunity to smallpox (in 1796), and by Pasteur's time this had generally replaced the use of actual smallpox material in inoculation. The difference between smallpox vaccination and anthrax or chicken cholera vaccination was that the weakened form of the latter two disease organisms had been generated artificially, and so a naturally weak form of the disease organism did not need to be found.

This discovery revolutionized work in infectious diseases, and Pasteur gave these artificially weakened diseases the generic name of vaccines, in honour of Jenner's discovery. Pasteur produced the first vaccine for rabies by growing the virus in rabbits, and then weakening it by drying the affected nerve tissue.

The rabies vaccine was initially created by Emile Roux, a French doctor and a colleague of Pasteur who had been working with a killed vaccine produced by desiccating the spinal cords of infected rabbits. The vaccine had only been tested on eleven dogs before its first human trial.

This vaccine was first used on 9 year old Joseph Meister, on July 6, 1885, after the boy was badly mauled by a rabid dog. This was done at some personal risk for Pasteur, since he was not a licensed physician and could have faced prosecution for treating the boy. After consulting with colleagues, Pasteur decided to go ahead with the treatment. Meister did not contract the disease. It is sometimes said that Pasteur saved the boy's life; but this cannot be maintained with certainty, since the risk of contracting rabies after such an exposure is estimated at around 15%. Nonetheless, Pasteur was hailed as a hero and the legal matter was not pursued. The treatment's success laid the foundations for the manufacture of many other vaccines. The first of the Pasteur Institutes was also built on the basis of this achievement.

Legal risk was not the only kind Pasteur undertook. In The Story of San Michele, Axel Munthe writes of the rabies vaccine research: