After early roles in the films Troop Beverly Hills (1989), This Boy's Life (1993), Son in Law (1993), and Snake Eyes (1998), Gugino received wider recognition for her starring roles in the Spy Kids trilogy (2001–2003), Sin City (2005), Night at the Museum (2006), American Gangster (2007), Righteous Kill (2008), Race to Witch Mountain (2009), Sally Jupiter in Watchmen (2009), Sucker Punch (2011), Mr. Popper's Penguins (2011), San Andreas (2015), Gerald's Game (2017), Gunpowder Milkshake (2021), and Lisa Frankenstein (2024).

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.

[2] 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 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.

Gugino made her Broadway debut in the Roundabout Theatre Company production of Arthur Miller's play, After the Fall opposite Peter Krause.

The following year, she played the female lead in the thriller Righteous Kill, opposite Robert De Niro and Al Pacino.

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.

During the Foundation's 34th Anniversary Gala in Washington, D.C., she received NIAF's Special Achievement Award for Entertainment, presented by her close friend, actress Connie Britton.