Carla Gugino was born in Sarasota, Florida, to Carl Gugino, an orthodontist of Italian descent, and a mother of English-Irish descent, described as "Bohemian".Her parents separated when she was two, after which she would travel between her father and half-brother Carl Jr.'s home in Sarasota, and her Paradise, California home, to which her mother moved her when she was four. She has described her bicoastal childhood, saying, "I lived in a tepee in Northern California and a van in Big Sur. With my dad, I lived in a beautiful house with a swimming pool and a tennis court and went to Europe for the summers. So I feel like I lived two childhoods." She worked as a teenage fashion model, and took acting classes at the suggestion of her aunt, former Let's Make a Deal spokesmodel Carol Merrill.She eventually came to support herself, and with her parents' support, was legally emancipated by the time she was 16.
Gugino is currently dating writer/director Sebastian Gutierrez, with whom she has worked on her TV series "Karen Sisco" and "Rise: Blood Hunter".
Gugino's television work during the late 1980s and early 1990s includes appearances on Saved by the Bell, Who's the Boss?, ALF, Doogie Howser, M.D. and The Wonder Years.
Gugino co-starred with Pauly Shore in the 1993 romantic comedy film Son in Law. She subsequently appeared in the video to Bon Jovi's 1994 song "Always".
After playing Michael J. Fox's love interest, Ashley Schaeffer, during the first season of the sitcom Spin City in 1996, Gugino appeared in the 1997 art-house film Lovelife. Her 1998 film work includes co-starring with Nicolas Cage in Brian De Palma's Snake Eyes, and appearing in Judas Kiss, which she also co-produced. She appeared as Dr. Gina Simon during the 1999–2000 final season of Chicago Hope.
In 2001, she appeared as family matriarch Ingrid Cortez in the first Spy Kids film by director Robert Rodriguez (as well as that film's two sequels in 2002 and 2003). That same year she appeared as Jet Li's love interest in the martial arts action thriller The One.
She starred in two short-lived TV series, the ABC crime drama Karen Sisco in 2003, and the CBS science fiction series Threshold in 2005 on CBS. That same year, Gugino appeared as Lucille in the feature film adaptation of Frank Miller's graphic novel, Sin City. The following year, she appeared in Night at the Museum.
Gugino appeared in the Roundabout Theatre Company play After the Fall opposite Six Feet Under's Peter Krause. In late 2006, she appeared in an Off-Broadway production of Tennessee Williams' Suddenly Last Summer opposite Blythe Danner.
Gugino appeared as Amanda, Vincent Chase's agent, in a dozen episodes of the cable television series Entourage. Gugino appeared nude in the May 2007 issue of Allure. That same year she appeared in the feature film American Gangster. The following year, she played the female lead in the thriller Righteous Kill, opposite Robert De Niro and Al Pacino.
Gugino starred in Chicago's Goodman Theater production of Eugene O'Neill's Desire Under the Elms from January 17 to February 17, 2009, in the role of Abby. Charles Isherwood of The New York Times praised Gugino's performance, saying, "Ms. Gugino displays a depth and range of expression that I cannot imagine any other actress achieving with such blazing honesty and wrenching truth. She is simply magnificent." During the first three months of 2009, three feature films premiered featuring Gugino: the thriller The Unborn, the film Watchmen, in which she played Sally Jupiter, and the adventure remake Race to Witch Mountain, in which she starred opposite Dwayne Johnson. That April, she received an Outer Critics Circle Award nomination for Outstanding Actress In a Play for her performance in Desire Under the Elms. Later in November of that year, she appeared as pornographic actress Elektra Luxx in the comedy film Women in Trouble.
In 2011 Gugino appeared as Madame Vera Gorsky in Zack Snyder's action-fantasy film Sucker Punch alongside Abbie Cornish and Emily Browning. Gugino sang a duet with co-star Oscar Isaac, which appeared in the end credits and in the film's soundtrack. She also played Henry "Hank" Moody's Attorney and Love-interest for the 4th season of Californication on Showtime.