Yeast EVs are a major pathway of non-classical protein secretion and have been implicated in basic cellular physiology, pathogenesis and hypervirulence. Proteomic characterization of yeast EVs revealed that many of the canonical biomarkers used in mammalian systems, including the highly conserved ESCRT proteins, are not present. However, S. cerevisiae strains with deletions in ESCRT proteins produce fewer EVs and these EVs have markedly different proteomic profiles to the wild type strain supporting a role for ESCRT in EV biogenesis in yeast. Isolation and proteomic characterization of EVs from three C. albicans strains grown in liquid culture, together with one strain grown as a biofilm, led to identification of 18 proteins that are potential EV markers. These included a subset of proteins with four transmembrane domains that are topologically similar to mammalian tetraspannins which are highly conserved EV markers in mammalian cells. These markers have application in the identification of EVs and tracking the EVs during purification or in a biological system such as an infection model. EV cargo also included proteins that may enhance resistance to antifungal drugs such as the efflux pumps Cdr1/2, the 1,3 β-glucan synthase Fks1 (as well as its regulator Rho1) and the major cell wall chitin synthase Chs3. We discovered that yeast EVs protect yeast against the antifungal activity of caspofungin and a plant antifungal defensin supporting a role for EVs in enhancing tolerance to antifungal molecules. We have also isolated and characterised EVs from Fusarium oxysporum and Fusarium graminearum filamentous fungal pathogens of plants. These EVs contain proteins with similar functions to those in yeast EVs in addition to proteins associated with virulence in the plant host. Understanding the role of EVs in the fungal plant interaction will identify new targets for more effective control of fungal diseases in agriculture and in the clinic.