Acker made her major television debut when she starred as Winifred "Fred" Burkle on the 2nd - 5th seasons of Angel, and also as the character of Illyria for part of the show's 5th and final season. She won the 2003 Saturn Award for Best Supporting Actress on Television for her portrayal.

She joined the cast of Alias in 2005 for its final season, playing the villainous character, Kelly Peyton. Acker graduated from guest star to fully credited cast member as the show entered the final episodes in April and May 2006.

Also in 2005, Acker provided voice acting for the character of Huntress on the animated series Justice League Unlimited.
She portrayed Dr. Claire Saunders/Whiskey, a recurring character on Joss Whedon's Dollhouse. Acker guest starred in 10 of the 13 episodes of the first season and three episodes of the second.

In 2010 she was a series regular in the ABC drama Happy Town, portraying the character Rachel Conroy. Also in 2010, she starred in the season-one finale of the Fox series Human Target, as the mysterious Katherine Walters. On May 25, 2010, she appeared on CBS's The Good Wife. She appeared in the horror movie The Cabin in the Woods, released April 13, 2012. She also portrayed Samantha "Root" Groves on CBS's Person of Interest in the season-1 finale/cliffhanger, and guest starred on multiple episodes on the subsequent season. Also in 2012, she revisited the science fiction and fantasy genres that had brought her to prominence, with guest performances in Warehouse 13, Once Upon A Time, and Grimm.