Leguizamo was born in Bogotá, Colombia. His father, Alberto Leguizamo, was a Puerto Rican Realtor living in Colombia who met and married a young Colombian woman by the name of Luz. In 1968 the couple immigrated with their children to the United States and settled down in the Jackson Heights section of Queens in New York City. Leguizamo received his primary and secondary education in Queens. As a student in high school, Leguizamo would write comical material and test it out on his classmates. He was voted "Most Talkative" by his fellow classmates. After graduating from high school (Murry Bergtraum High School), Leguizamo enrolled at New York University where he took theater classes.

Leguizamo started out as a stand-up comic doing the New York nightclub circuit. In 1984, he made his T.V. debut with a small part in Miami Vice. In 1985, Leguizamo landed a small uncredited part, making his movie debut, in Mixed Blood and in 1989 he also had a small part in Casualties of War. In 1991, Leguizamo participated in Hanging with the Homeboys. That same year, he wrote and participated in the Off-Broadway production Mambo Mouth, where he played seven different characters. Mambo Mouth won an Obie Award and an Outer Critics Award. He was listed as one of 12 "Promising New Actors of 1991" in "John Willis' Screen Worlds Vol. 43".

In 1993, Leguizamo wrote and participated in Spic-O-Rama, where he made fun of the stereotyping of Latinos in the U.S.. The production won a Drama Desk Award and four Cable ACE Awards. The 1993 movie production Super Mario Brothers where he played the role of "Luigi Mario", is considered to be the film which skyrocketed his acting career in Hollywood, even though the film bombed spectacularly. Since then, other movies followed, such as: Carlito's Way (1993) as "Benny Blanco" alongside Al Pacino, Romeo + Juliet (1996) as "Tybalt", The Fan (1996) as "Manny", Executive Decision (1996) as "Captain Rat", as con man Pestario "Pest" Vargas The Pest in 1997 in which he starred alongside his wife at one point, Yelba Osorio whom he later was divorced from, Doctor Dolittle (1998) as "Rat#2", Summer of Sam (1999) and Moulin Rouge! as "Toulouse Lautrec". In 2000, Leguizamo played two genies in the Emmy Award winning miniseries Arabian Nights. Leguizamo has participated in over 53 films, including The Alibi where he will play the role of "Hannibal" and which is in the post-production stages as of 2005.

Leguizamo also created, executive produced, wrote for, and starred in the 1995 Latino-oriented variety show called House of Buggin'. The show showcased Leguizamo's well-known ability to assume a wide variety of colorful, energetic characters. The show ran less than a year on Fox.

In 1998, he debuted on Broadway in the production of Freak, which was also turned into an HBO film by director Spike Lee. Leguizamo has produced 10 films, including Piñero and made 13 TV guest appearances.

Later, in 2003, he voice-acted Globox from Rayman 3. The game versions with his voice acting were PlayStation 2, GameCube, Xbox, PC, and Mac.

In 2005, Leguizamo joined the cast of the show ER, playing Dr. Victor Clemente, a new attending who is keen on introducing the staff of County General to ways of treating patients and cutting-edge technology.