After completing high school, Dushku returned to acting with the role of Faith on Buffy the Vampire Slayer, a Slayer much more troubled than the main character Buffy Summers. Though initially planned as a five episode role, the character became so popular that she stayed on for the entirety of the third season and returned for a two-part appearance in season four, after which the remainder of her original story arc was played out as part of the first season of the Buffy spin-off series Angel. Repentant and rededicated, Faith returned as a heroine in a number of further episodes of Angel and in the last five episodes of Buffy. Dushku was inundated with piles of fan mail from legions of prisoners. She said that:
I've been getting fan mail from maximum security penitentiaries and death row. What are the authorities thinking of in playing a show with young teenage girls to Death Row inmates? They write everything — disgusting things that you don't even want to know about. And they send me pictures — 'Oh, here's a picture of me before I was incarcerated!' — and there's some guy sat on the sofa with a bottle of beer and a moustache, and a big gut. It's so creepy. Way more creepy than Buffy.
In 2000, Dushku starred in Soul Survivors, reuniting her with Race The Sun co-star Casey Affleck. She followed that up with the cheerleader comedy Bring It On with Kirsten Dunst, which was a surprising success at the box office that spawned straight-to-DVD sequels. In 2001, she appeared in The New Guy with DJ Qualls and City by the Sea with Robert De Niro and James Franco. The latter film garnered attention from a wider adult audience and several good reviews.
The same year, Kevin Smith invited Dushku to be a part of Jay and Silent Bob Strike Back, where she co-starred with Shannon Elizabeth, Ali Larter, and Ben Affleck.
In 2003, Dushku appeared in Wrong Turn, a horror film in which she had the starring role, and The Kiss, an independent comedy-drama. Starting that same year, she also starred in a new Fox TV series, Tru Calling, where she played the main character, medical student Tru Davies. After having a grant pulled out from under her, Tru is forced to take a job at a local morgue where she discovers her power to "re-live" the previous day over again and uses it to bring murderers to justice. Dushku turned down a role in a spin-off of Buffy The Vampire Slayer (that would have been about Faith).
She has had many roles as a "bad girl" in movies and relishes the opportunities. In an interview with Maxim in May 2001, Dushku says of her roles, "It’s easy to play a bad girl: You just do everything you’ve been told not to do, and you don’t have to deal with the consequences, because it’s only acting."
Dushku starred in an off-Broadway production entitled Dog Sees God from December 2005, playing "Van's sister", a character paralleled with Lucy from the original Peanuts comic strip on which the play production is based. She quit in February 2006 along with several other members of the cast among rumours of alleged abuse from the producer, which were later dismissed.
She played the lead character on Nurses, a hospital comedy/drama for Fox. This was the second Fox pilot in which she has been cast, but will not be broadcast. She appeared in the Simple Plan music video, "I'm Just a Kid", as the band's love interest, as well as Nickelback's video for "Rockstar".
Dushku has landed starring roles in two video game productions. She voiced the role of Yumi Sawamura in the English language version of Yakuza for the PlayStation 2, which was published and developed by SEGA, and released in September 2006. Dushku also stars as Shaundi, one of the lead characters in Saints Row 2, which was developed by Volition and published by THQ. It was released (in North America) on October 14, 2008 for the PlayStation 3 and Xbox 360.