In 2001, she appeared in the independent film Question of Faith, where she played the monk Anselm. Her character underwent a sex change from male to female as part of a Divine miracle. The film was directed by her husband.

She was cast in Star Trek: Voyager in the recurring role of Seska, a Bajoran member of the Maquis crew who joined Voyager after it was stranded in the Delta Quadrant. She first appeared in the episode "Parallax" where she wore the blue uniform of a Starfleet science officer; this was later revealed to be a costuming error and she was subsequently seen in the uniform of the operations department. The original uniform sold for $1,276 ($1,450 today) in the "It's a Wrap!" auction on eBay in 2008.

When she was first cast in the role of Seska, she was not informed that she was a Cardassian spy. She later explained that "when I first started I was Seska the Bajoran Maquis member and they sort of worked everything else up in soap opera fashion." Hackett became pregnant, which was written into Seska's character. Hackett described Seska as being a character with several layers, saying that "Seska’s darker sides came from a certain emotional instability, at least that’s how I played her." Seska was killed off in the second part of "Basics", which came as a surprise to Hackett as she was previously sent a version of the script where Seska survived, but her baby died. She was only told that Seska was to die less than a day before filming. Following her character's death, Hackett as Seska returned to Voyager twice more in the episodes "Worst Case Scenario" and "Shattered". She appeared in a total of thirteen episodes of Voyager.