Extracellular vesicle delivery in Candida biofilms is important for matrix delivery and associated drug-resistance. Prior study found the antifungal turbinmicin inhibits vesicle release. We find the antifungal turbinmicin inhibits both vesicle release as well as cargo loading. Using comparative vesicle proteome analysis in turbinmicin treated biofilms we identify several components blocked from cargo loading that play specific roles in the functional matrix protection mechanism.