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Summary  In winemaking, the use of alternative yeast starters is becoming increasingly popular. They contribute to the diversity and complexity of wine sensory features and are typically used in combination with  Saccharomyces cerevisiae , to ensure complete fermentation. This practice has drawn the interest on interactions between different oenological yeasts, which are also relevant in spontaneous and conventional fermentations, or in the vineyard. Although several interactions have been described and some mechanisms have been suggested, the possible involvement of extracellular vesicles (EVs) has not yet been considered. This work describes the production of EVs by six wine yeast species ( S. cerevisiae ,  Torulaspora delbrueckii ,  Lachancea thermotolerans ,  Hanseniaspora uvarum ,  Candida sake  and  Metschnikowia pulcherrima ) in synthetic grape must. Proteomic analysis of EV‐enriched fractions from  S. cerevisiae  and  T. delbrueckii  showed enrichment in glycolytic enzymes and cell‐wall‐related proteins. The most abundant protein found in  S. cerevisiae ,  T. delbrueckii  and  L. thermotolerans  EV‐enriched fractions was the enzyme exo‐1,3‐β‐glucanase. However, this protein was not involved in the here‐observed negative impact of  T. delbrueckii  extracellular fractions on the growth of other yeast species. These findings suggest that EVs may play a role in fungal interactions during wine fermentation and other aspects of wine yeast biology.

Proteomic characterization of extracellular vesicles produced by several wine yeast species