Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. is a eukaryotic gene that is required for the activity of benzimidazoles against . In this study, during the phenotypic characterization of mutants expected to lack expression, we observed a reduced EV production. Whole-genome sequencing, RNA-Seq, and cellular proteomics revealed that, contrary to our initial findings, these mutants expressed Nop16 but exhibited altered expression of 14 genes potentially involved in sugar transport. Based on this observation, we designated these mutant strains as Past1 and Past2, representing otentially ltered ugar ransport. Analysis of the small molecule composition of EVs produced by wild-type cells and the Past1 and Past2 mutant strains revealed not only a reduced number of EVs but also an altered small molecule composition. In a model of infection, the Past1 and Past2 mutant strains were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the mutant cells in . These results connect EV biogenesis, cargo, and cryptococcal virulence.