Lyonne began acting at an early age with her first role as “Opal” on Pee-wee's Playhouse. She appeared in the 1990 film A Man Called Sarge during the time her family lived in Israel.

Lyonne is perhaps best known for her roles in the first two American Pie films as the wise-cracking Jessica. She has also appeared in more than 30 other movies, including starring roles in the independent films Slums of Beverly Hills and But I'm a Cheerleader. Lyonne also appeared in Party Monster in 2003, a dramatization of the Michael Alig story. As the character Brooke, Lyonne donned a fat suit and dreadlocks. She also played a blind biologist in Blade: Trinity.

After an extended hiatus, Lyonne filmed Goyband in New York City with Adam Pascal, Amy Davidson, Cris Judd, Dean Edwards, Tibor Feldman, and Tovah Feldshuh.

In January 2008, she appeared in an Off-Broadway play, Two Thousand Years. In November 2010 she appeared as “Grace” in the play Tigers Be Still at the Roundabout Theatre Company.

Lyonne worked on the thriller 13 with a release date of 2010. Other cast members include Mickey Rourke, Ray Liotta and Jason Statham.

Lyonne is currently filming the dark comedy/horror film All About Evil in San Francisco. The film was written by and directed by San Francisco late-night movie host and short film producer Joshua Grannell.