MacNeille worked in a variety of jobs and had numerous minor voiceover roles before becoming a regular on an animated TV show. In her words, "I'd been doing radio spots, some TV, demos, sound-alikes, industrial narrations -- anything that came my way for about two years." She was also a member of the improvisational comedy group The Groundlings for ten years. MacNeille took acting workshops and worked as a casting assistant for voice acting talent agent Bob Lloyd in what she calls "The University of Voice-over." Lloyd and fellow agent Rita Vennari got MacNeille her first role on an animated show: a part in an episode of the 1979 Scooby-Doo and Scrappy-Doo.
She sang and appeared in the music video (as Lucille Ball) for "Weird Al" Yankovic's song "Ricky" (1983), which was based on the I Love Lucy television show and parodied the song "Mickey" by Toni Basil. MacNeille also appeared on Yankovic's 1999 album Running with Scissors, on the tracks "Pretty Fly for a Rabbi" and "Jerry Springer."
MacNeille was cast as Babs Bunny in Tiny Toon Adventures (1990–1995). Writer Paul Dini said that MacNeille was good for the role because she could do both Babs' voice and the voices of her impressions. MacNeille commented: "The best part of doing Babs is that she's a mimic, like me...In the show I do Babs doing Billie Burke, Hepburn, Bette Davis, Madonna and Cher. I even have her doing Jessica Rabbit." The success of Tiny Toon Adventures led to the series Animaniacs. MacNeille was brought in to voice Dot Warner, one of the show's three main characters, because Dot's character was very similar to Babs Bunny. Andrea Romano, the voice Director and caster for Animaniacs, said that the casters had "no trouble" choosing the role of Dot: "Tress MacNeille was just hilarious (...) And yet [she had] that edge." MacNeille was nominated for an Annie Award for her performance on the show in 1995.
She is the current voice of Daisy Duck and Wilma Flintstone. MacNeille also appeared as an angry anchorwoman in Elvira, Mistress of the Dark and served as the voice of Elvira's Great-Aunt Morganna Talbot. She provided voice acting for the 2003 Wile E. Coyote and The Road Runner short feature The Whizzard of Ow.
