Gugino has starred in many films including art-house film Lovelife along with Saffron Burrows and 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). She played Ashley Schaeffer in the first season of the Michael J. Fox television series Spin City. She also starred in the short lived CBS science fiction series Threshold and in ABC's Karen Sisco, in which she recreated the law-enforcement role of Jennifer Lopez from the film Out of Sight.

Gugino appeared in Roundabout Theatre Company's After the Fall opposite Six Feet Under's Peter Krause. In late 2006, she will begin the Off-Broadway play by Tennessee Williams, Suddenly Last Summer opposite actress Blythe Danner. 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.

Gugino played Vincent Chase's new agent, Amanda, in the second half of Entourage's third season for a couple of episodes and will reprise that role in the fifth season. Gugino appeared nude in the May 2007 issue of Allure. She played Sally Jupiter, the original Silk Spectre, in the Watchmen movie, and had roles in The Unborn and Race to Witch Mountain (opposite Dwayne Johnson). She played the female lead in the serial killer thriller Righteous Kill opposite Robert De Niro and Al Pacino. She also stars in a new film directed by Gutierrez, Women in Trouble, as a pornographic actress named Elektra Luxx. Gugino starred in Chicago's Goodman Theater production of Eugene O'Neill's Desire Under the Elms January 17-February 17, 2009 in the role of Abby. The New York Times lauds, ""She [Gugino] is simply magnificent...it is impossible to look away."

• Elektra Luxx (2010) (filming) .... Elektra Luxx
• Every Day (2009) (post-production) .... Robin
• Women in Trouble (2009) (completed) .... Elektra Luxx
• Our Lady of Victory (2008) (completed) .... Cathy Rush
  
• Under the Hood (2009) (V) .... Sally Jupiter / Silk Spectre
  ... aka The Culpeper Minute: Under the Hood (UK: long title)
• Race to Witch Mountain (2009) .... Dr. Alex Friedman
• Watchmen (2009) .... Sally Jupiter / Silk Spectre
  ... aka Watchmen: The IMAX Experience (USA: IMAX version)
• Sparks (2009) .... Robin
• The Unborn (2009) .... Janet Beldon
• "Entourage" .... Amanda Daniels (7 episodes, 2007-2008)
       - First Class Jerk (2008) TV episode .... Amanda Daniels
       - Unlike a Virgin (2008) TV episode .... Amanda Daniels
       - The Return of the King (2007) TV episode .... Amanda Daniels
       - Gotcha! (2007) TV episode .... Amanda Daniels
       - Manic Monday (2007) TV episode .... Amanda Daniels
        (2 more)
• Righteous Kill (2008) .... Karen Corelli
• American Gangster (2007) .... Laurie Roberts
• Rise (2007) .... Eve
  ... aka Rise: Blood Hunter (USA: DVD title)
• The Lookout (2007) .... Janet
• Night at the Museum (2006) .... Rebecca
  ... aka Night at the Museum: The IMAX Experience (USA: IMAX version)
• Even Money (2006/I) .... Veronica
• "Threshold" .... Dr. Molly Anne Caffrey (13 episodes, 2005-2006)
       - Alienville (2006) TV episode .... Dr. Molly Anne Caffrey
       - Vigilante (2006) TV episode .... Dr. Molly Anne Caffrey
       - Outbreak (2006) TV episode .... Dr. Molly Anne Caffrey
       - The Crossing (2006) TV episode .... Dr. Molly Anne Caffrey
       - Progeny (2005) TV episode .... Dr. Molly Anne Caffrey
        (8 more)
• Sin City (2005) .... Lucille
  ... aka Frank Miller's Sin City (USA: complete title)