Gugino was born in Sarasota, Florida, of Italian, English, and Irish ancestry. Her parents separated when she was two and 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 graduated 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 catwalk work. Finding the experience too much, Gugino moved back to California that summer. Partly at the suggestion of her aunt, game show model Carol Merrill, she subsequently studied acting.

Gugino has starred in many films including art-house film Lovelife along with Saffron Burrowsand 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).

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" and also on the last season of Chicago Hope as brain surgeon Dr Gina Simon. Gugino also frequently appears in films written and directed by her longtime boyfriend Sebastian Gutierrez, as they've worked together on several films in the past. Their latest collaboration is the vampire horror film Rise: Blood Hunter opposite Lucy Liu The film was in limited release beginning June 1, 2007.

Gugino played Vincent Chase's new agent, Amanda, in the second half of Entourage's third season for a couple of episodes. Gugino appeared nude in the May 2007 issue of Allure. She will play Sally Jupiter, the original Silk Spectre, in the upcoming Watchmen movie.