Born in Sarasota, Florida to an Italian-Irish father and a mother of English descent. Her mother moved the family to Paradise, California, when she was four years old. Despite moving around California a great deal as a child, Carla Gugino remained a straight-A student and graduate valedictorian. At the age of 15 she was spotted by a modeling agency in San Diego and moved to New York. This was unusual as at only 5 feet 4 inches (1.65 meters) tall, she was considered too short for runway work. Finding the experience too much, Gugino moved back to California that summer. She subsequently studied acting.

Carla Gugino has appeared in many films including Snake Eyes (1998), Judas Kiss (which she also co-produced, 1998), the three Spy Kids films, Son in Law, and most recently as a lesbian parole officer in Sin City. Carla Gugino has also appeared in a wide range of TV programs including ALF, Doogie Howser, M.D., Saved by the Bell, Who's the Boss? and The Wonder Years, and is featured in the video to Bon Jovi's 1994 song 'Always'.

She also played Ashley Schaeffer in the television series Spin City. Gossip suggested Gugino was removed after twelve episodes because Tracy Pollan—wife of star Michael J Fox—felt her chemistry with Fox was too good.