Machado later starred in the short-lived series Missing, Three Rivers and Welcome to the Family and was a regular cast member in the first season of the USA Network crime drama Queen of the South.

Machado was a guest appearing in Episode 6 of Season 6 of Finding Your Roots, a documentary show by PBS that is hosted by Dr. Henry Louis Gates, Jr., during which it was confirmed that she has African matrilineal ancestry (i.e. through a female ancestor on her mother's side[7]) through genetic testing of her mitochondrial DNA.

She was cast as Elsa in the ABC series NYPD Blue appearing in an episode "Burnin' Love", and co-starring in the made-for-television drama film No One Would Tell.

[2] In 1997, she made her big screen debut playing supporting role of Carmen Rodriguez in the romantic drama film She's So Lovely.

[3] In 2003, she played Isabella Hudson in the supernatural horror film, Final Destination 2, and the following year appeared in the action comedy Torque.

In 2003, Machado appeared in the music video for TLC's song "Damaged", the third single from their fourth studio album 3D, as a young woman trapped in an abusive relationship and unsure of what to do with her life.

[2] From 2003 to 2004, Machado also starred in the Canadian crime drama series, 1-800-Missing playing FBI Agent Sunny Estrada during the show first season.

Machado has also made appearances on stage, including a 2008 production of Neil LaBute's play, Some Girl(s), at the Geffen Playhouse in Los Angeles.

[4][14] Machado has received positive reviews for her role as a single mother raising two children and struggling with PTSD after serving in Afghanistan.

[19] From 2019 to 2020, she had a recurring role on the NBC comedy series, Superstore playing new district manager Maya, with whom Amy Sosa shares a surprising connection.

[20] After having been announced to portray Florence Johnston in the first Live in Front of a Studio Audience’s The Jeffersons portion, instead reprised by Marla Gibbs,[21] Machado was cast in the second edition in December 2019 as Teresa Betancourt from All in the Family.

[22] On September 2, 2020, Machado was announced as one of the celebrities competing on the 29th season of Dancing with the Stars, partnered with professional dancer, Sasha Farber.

Also in 2020, Jacob Vargas and Justina Machado were Masters of Ceremonies at the National Hispanic Media Coalition Impact Awards.