Species of the genus Paracoccidioides cause a systemic infection in human patients. Yeast cells of Paracoccidioides spp. produce melanin in the presence of L-dihydroxyphenylalanine and during infection, which may impact the pathogen survival into the host. To better understand the metabolic changes that occur in melanized Paracoccidioides spp. cells, a proteomic approach was performed to compare melanized and non-melanized Paracoccidioides brasiliensis and Paracoccidioides lutzii yeast cells. Melanization was conducted using L-dihydroxyphenylalanine as a precursor and quantitative proteomics was performed using reversed-phase chromatography coupled to high resolution mass spectrometry. When comparing melanized versus non-melanized cells, 999 and 577 differentially abundant proteins were identified for P. brasiliensis and P. lutzii, respectively. Functional enrichment and comparative analysis revealed 30 abundant biological processes in melanized P. brasiliensis and 18 in P. lutzii, while non-melanized cells from these species had 21 and 25 differentially abundant processes, respectively. Melanized cells presented abundance of other virulence-associated proteins, such as phospholipase, proteases, superoxide dismutase, heat-shock proteins, as well as proteins related to cell-wall remodeling and vesicular transport. The results suggest that L-dihydroxyphenilalanine increases virulence of Paracoccidioides spp. through a complex mechanism involving not only melanin, but other virulence factors as well.