Gugino has starred in many films including art-house film Lovelife along with Saffron Burrows and Sherilyn Fenn, Snake Eyes with Nicolas Cage (1998), Judas Kiss (which she also co-produced, 1998), the three Spy Kids films, Son in Law, The One with Jet Li, and Sin City. Her most recent roles came in box office hits such as Night at the Museum (2006) and American Gangster (2007).

She played Ashley Schaeffer in the first season of the Michael J. Fox television series Spin City. She also starred in the short lived CBS science fiction series, Threshold and in ABC's Karen Sisco, in which she recreated the law-enforcement role of Jennifer Lopez from the film Out of Sight

Gugino appeared in Roundabout Theatre Company's After the Fall opposite Six Feet Under's Peter Krause. In late 2006, she will begin the Off-Broadway play by Tennessee Williams, Suddenly Last Summer opposite actress Blythe Danner.

Gugino has also appeared in a wide range of TV programs including ALF, Doogi