Flores was born in Cotió, Mixco, a town near Guatemala City. During his early athletic career, Flores worked in as a low-paid laborer in a textile factory. He would wake at 4:00 am to run for two hours, and run an additional two hours after returning from work at 6:00 pm.
On April 19, 1952, Flores participated in the Boston Marathon, a major international racing event. He took the lead from his countryman Luis H. Velasquez after the first ten miles, and, against any predictions, finished ahead of U.S. competitor Victor Dyrgall by almost five minutes, recording a time of 2 hours, 31 minutes, and 53 seconds.His record remained as a national record for Guatemala for seventeen years, until November 5, 1969, when it was broken by Julio Quevedo.
The Boston achievement made Flores a sports icon in his country, and the Guatemalan government paid him tribute by renaming the national stadium in Guatemala City to Estadio Nacional Mateo Flores. He was treated as a national hero by the Guatemalan government, which also awarded him the Order of the Quetzal (the nation's highest civilian honor).