Her earliest known work, " Monkey Child" , was painted in 1575 at the age of 23. Though this work is now lost, another early painting, Christ with the Symbols of the Passion, painted in 1576 is now in the El Paso Museum of Art. She would go on to paint in a variety of genres. Early in her career, she was most famous for painting upper-class residents of her native Bologna. She began her commercial practice by painting small devotional paintings on copper, which had popular appeal as papal and diplomatic gifts, given the value and lustre of the metal. She later created paintings of male and female nudes and large scale religious paintings.

Fontana married Paolo Zappi (alternately spelled Paolo Fappi) in 1577. She gave birth to 11 children, though only 3 outlived her. After marriage, Fontana continued to paint to support her family. Zappi took care of the household and served as painting assistant to his wife, including painting minor elements of paintings like draperies.

Fontana and her family moved to Rome in 1603 at the invitation of Pope Clement VIII. She gained the patronage of the Buoncompagni, of which Pope Gregory XIII was a member. Lavinia thrived in Rome as she had in Bologna and Pope Paul V himself was among her sitters.

Some of her portraits, often lavishly paid for, have been wrongly attributed to Guido Reni. Chief among these are Venus; The Virgin lifting a veil from the sleeping infant Christ; and the Queen of Sheba visiting Solomon. Her self-portrait – in youth she was said to have been very beautiful – was perhaps her masterpiece; it belongs to Count Zappi of Imola, the family into which Lavinia married.

While her youthful style was much like her father's, she gradually adopted the Carracciesque style, with strong quasi-Venetian coloring. She was elected into the Accademia di San Luca of Rome, and died in that city on August 11, 1614.

There are over 100 works that are documented, but only 32 signed and dated works are known today. There are 25 more that can be attributed to her, making hers the largest oeuvre for any female artist prior to 1700. Sofonisba Anguissola may have been an influence on her career.