By 1991, Darbo had featured in numerous stage, film and television credits and was contracted to the ABC network. In 1991, the Los Angeles Times reported that Darbo was undertaking a personal campaign to seek nomination for an Academy Award for Best Supporting Actress for her role in Daddy's Dyin': Who's Got the Will?, written by Del Shores.
Darbo appeared as Penny Baker on Step by Step (1991–1992). In 1992 she appeared in the feature film Leaving Normal under the name 66 with Meg Tilly and Christine Lahti. In 1994, she starred as Roseanne Barr in an NBC television movie, Roseanne and Tom: Behind the Scenes. She starred as Miss Spencer in Ruby Bridges (1998). From 1998 to 2005, Darbo played Nancy Wesley on the NBC soap opera Days of Our Lives, a role that earned her an Emmy nomination for outstanding supporting actress in 2000.
Darbo was a guest star in two Seinfeld episodes; "The Revenge" (1991) and "The Sniffing Accountant" (1993). Later, in 2010, she also guest starred in Showtime's Dexter as a landlady. Between 2010 and 2012 she portrayed the recurring role of Dr. Freed in the teen drama web series Miss Behave.
In 2012, Darbo played Grams in Mickey Matson and the Copperhead Conspiracy. In November 2012, it was announced that Darbo would reprise her role as Nancy Wesley on Days of Our Lives in 2013. After a three-year absence, Darbo returned to the series in the fall of 2016.
In 2016, Darbo won an Emmy for Best Actress in a Short Form Comedy or Drama Series for her work in the series Acting Dead. She is the first winner of this category.