MALDI-TOF MS mass spectrometry is a rapid and straightforward technique to identify microorganisms by protein analysis. The study was performed in 304 yeast isolates from superficial and deep mycoses, in order to compare three methods: conventional (biochemical and morphological), MALDI-TOF MS, and polymerase chain reaction (PCR, reference). We included 24 species with predominance of Candida spp and Cryptococcus spp. The identification by conventional methods was 258/304 strains, while by MALDI-TOF MS was: 277/304 strains (84.8% versus 91.2%, P = not significant). The Kappa coefficient comparing MALDI-TOF-MS with PCR reported excellent concordance (0.99). The sensitivity and specificity of MALDI-TOF MS for the diagnosis of opportunistic pathogenic yeasts of clinical samples were 94.6% and 99% respectively. MALDI-TOF MS is a simple, fast and reliable tool for pathogenic yeasts.