Mastroeni moved to the United States from Argentina with his family at the age of four, settling in Phoenix, Arizona.

Mastroeni attended North Carolina State University where he played on the men's soccer team from 1994 to 1997.

The Fusion was contracted after the 2001 season and Mastroeni was the first overall choice of the 2002 MLS Allocation Draft, going to the Colorado Rapids, whom he captained to their first title in 2010.

When Chris Armas went out with an injury just weeks prior to the 2002 World Cup, Mastroeni found himself a starter in the opening game as the U.S. defeated Portugal 3–2.

In a FIFA World Cup 2006 match against Italy on June 17, Mastroeni was given a red card for an aggressive tackle in the first half.

Mastroeni was named the interim head coach of the Colorado Rapids after Oscar Pareja left for FC Dallas in January 2014.

The Rapids ended the season last in the Western Conference and second to last in the overall standings, receiving the second overall pick in the upcoming MLS Superdraft.

During the offseason before the 2016 season, Mastroeni visited London, spending several days watching Arsenal and Tottenham train.