Gugino’s television work during the late 1980s and early 1990s included appearances on Good Morning, Miss Bliss, Saved by the Bell, Who’s the Boss?, ALF, Doogie Howser, M.D., The Wonder Years, Webster and a recurring role on Falcon Crest. In film, Gugino appeared in the Shelley Long film Troop Beverly Hills, and she co-starred with Pauly Shore in the romantic comedy Son in Law

In 1995, Gugino appeared as Nan St. George (later the Duchess of Trevenick) with Greg Wise and James Frain in the BBC miniseries The Buccaneers, an adaptation of Edith Wharton’s last novel. She played Ashley Schaeffer, Michael J. Fox’s character’s love interest, during the first season of the sitcom Spin City in 1996. She played opposite Nicolas Cage in Brian De Palma’s Snake Eyes, and in Judas Kiss, which she also co-produced. She appeared as Dr. Gina Simon during the final season of the television medical drama Chicago Hope (1999–2000).

In 2001, she appeared as family matriarch Ingrid Cortez in the first Spy Kids film (as well as the 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: ABC’s Elmore Leonard crime drama Karen Sisco in 2003, and CBS’ science fiction series Threshold in 2005. 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 the film Night at the Museum as Ben Stiller’s love interest.

Gugino appeared as Amanda, Vincent Chase’s agent, in a dozen episodes of the cable television series Entourage. Gugino appeared in the May 2007 issue of Allure. That same year she appeared in the action-horror film Rise: Blood Hunter and 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.

In 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. Later, in November of that year, she played a pornographic actress in the comedy film Women in Trouble, which spawned a sequel in 2010, Elektra Luxx, titled after her character.

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 guest starred on the fourth season of Californication as Abby Rhodes, Hank Moody’s attorney and love interest.

In 2012, Gugino had a lead role as Susan Berg, a Washington, D.C., investigative reporter, on the USA Network’s miniseries Political Animals. In 2015, she had a lead role in the disaster film San Andreas, in which she once again starred opposite Dwayne Johnson.

She has also appeared in Netflix’s The Haunting of Hill House, The Haunting of Bly Manor, and The Fall of the House of Usher. Jost recently she appeared in The Girls on the Bus and Lisa Frankenstein.