AbstractPolymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human pathogenic fungusAspergillus fumigatustriggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. Two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae, which led to reduced fungal growth relative to a control retinol binding protein 7 producing strain. In conclusion, the production of afEVs by PMNs offers an intriguing, previously overlooked mechanism of antifungal defense againstA. fumigatus.ImportanceInvasive fungal infections caused by the moldAspergillus fumigatusare a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high mortality as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination ofA. fumigatusfrom the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils againstA. fumigatusnot previously described. We find that extracellular vesicles produced by neutrophils in response toA. fumigatusinfection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding ofA. fumigatushost pathogenesis and opens up novel diagnostic and therapeutic possibilities.