Beginning her career on the soap opera One Life to Live, Aylesworth also had minor roles in Random Hearts, and You've Got Mail. After guest starring roles in Law & Order, Law & Order: Special Victims Unit, Now and Again, and The West Wing, Aylesworth was part of the 2001 series All Souls, which was cancelled during its first season. In 2002, Aylesworth joined the cast of another series, American Embassy which also failed to gain an audience.

In 2001, Aylesworth had auditioned for the part of Nina Myers on FOX Network's 24 but lost to Sarah Clarke. In 2002, she auditioned once again, this time for the role of Kate Warner, but lost to Sarah Wynter. The producers, however, asked her to join the cast as recurring character Michelle Dessler, a CTU agent.

Her character quickly developed a relationship with fellow agent Tony Almeida (Carlos Bernard), and became a firm fan favorite. By the third season, Aylesworth had become a main cast member married to Bernard's character. By the end of that season, the writers decided to have major cast changes, including the departure of her character. However, fan forums lit up with people asking for her to stay. In the twelfth episode of the fourth season, Aylesworth returned - now as a high-ranking bureaucrat in the organization and remained until the end of the season. However, her character was killed in the first episode of season 5.

Aylesworth also had a much-publicized guest starring role on CSI which was initially rumored to be a regular role. However, after fired cast members Jorja Fox and George Eads were rehired, and with the offer to return to 24, Aylesworth's role lasted only one episode.

Since her departure from 24, Aylesworth has guest starred on the short lived series Conviction, appeared on stage in The Fearless, filmed a supporting role in Kevin Costner’s Mr. Brooks, and has starred in the independent thriller The Killing Floor. Aylesworth was also originally cast as Dr. Adrienne Holland in the series 3 lbs. However, 3 lbs was not picked up at the time. When CBS finally decided to pick up the series for a late fall 2006 start, the leading roles were all recast. It is not known if Aylesworth simply declined to star in the new series due to commitments elsewhere or if she was not offered the role again.

On 25 September 2006 it was announced that Aylesworth was chosen for one of the two lead roles in Aliens vs. Predator: Requiem. She has been cast as Kelly O'Brien, a pilot and soldier. In January 2007, Aylesworth had a guest role in two episodes of the new ABC comedy, The Knights of Prosperity in which she played the head of Mick Jagger’s security company.