Justina Milagros Machado was born on September 6, 1972, in Chicago, Illinois, to Alicia Morales Ruiz and Ismael Machado. Her parents, both of Puerto Rican descent, divorced when she was young, and her mother remarried, giving Justina three half-siblings.

Growing up in a modest household on the Northwest Side of Chicago, Justina was raised Catholic and had a traditional quinceañera. She was very active in drama and dance during her school years, participating in school plays and taking dance classes at Franklin Fine Arts Center.

After graduating from Lane Technical College Prep High School in 1990, she moved to New York City to pursue a career in acting.

Her career began with secondary roles on television and film. Her breakthrough came in 2001 when she was cast as Vanessa Diaz in the HBO comedy-drama series "Six Feet Under." Her performance earned her critical acclaim and a Screen Actors Guild Award for Outstanding Performance by an Ensemble in a Drama Series.

She 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."

From 2017 to 2020, she starred as Penelope Alvarez in the Netflix/Pop TV comedy series "One Day at a Time," for which she received positive reviews from critics and two Imagen Awards for Best Actress - Television, as well as a nomination for the Critics' Choice Television Award for Best Actress in a Comedy Series.

Justina Machado has also appeared in several films, including "Final Destination 2" (2003), "Torque" (2004), "Little Fugitive" (2006), "The Call" (2013), "The Purge: Anarchy" (2014), "All Together Now" (2020), and "The Throwback" (2024).

In 2023, she played the leading role in the Amazon Prime Video black comedy-horror series "The Horror of Dolores Roach."