A native of Sarasota, FL, Gugino spent most of her youth being shuttled by her mother to various locations in California. At age 15, she and her mom were living in San Diego, when Gugino was convinced to try a modeling career in New York City. She succeeded, but soon found the fast pace of high-fashion modeling too much for her and moved to Los Angeles, where she took advice from an aunt, model Carol Merril, and enrolled in acting classes to study under drama coach Gene Bua. Gugino soon made her film debut in the comedy Troop Beverly Hills (1986).
More features followed, until she got her first supporting major role, that of Norma in the Robert De Niro/Leonardo Di Caprio drama This Boy's Life (1993), and later scored a co-starring role on the Michael J. Fox sitcom Spin City. As her star continued to rise, Gugino would spend the subsuquent years appearing in a wide variety of high profile projects, like Spy Kids, Out of Sight, Sin City, Night at the Museum, Sucker Punch, Watchmen, and Mr. Popper's Penguins. Gugino would also find major success on the small screen, with roles on shows like Entourage and Californication.