Extracellular vesicles (EVs) are membranous vesicles released into the extracellular environment by cells. EVs contain various functional cargo that play crucial roles in intercellular communications. In mammalian studies, it is well established that the Endosomal Sorting Complex Required for Transport (ESCRT) machinery is responsible for the biogenesis of exosomes, an important subtype of EVs with endocytic origin. The ESCRT machinery was first described for its involvement in endosome formation using yeast as a model. Mechanisms of yeast EV biogenesis and release remain unclear, thus further investigations on the characterization of yeast EVs are required. To understand more about the role of ESCRT proteins in the biogenesis of yeast EVs, we investigated the release of EVs by model yeast Saccharomyces cerevisiae upon knockout of selected ESCRT components. It was revealed by corresponding assays that EVs isolated from ESCRT knockout cells were altered in terms of protein amounts, protein cargo, morphologies as well as size compared to wild type cells. These differences are especially prominent in EVs upon knockouts of three Vps proteins. We also characterized different types of EVs from wild type yeast cultures upon specific drug treatments or gene deletions by proteomic analysis. The analysis highlighted the enrichment of functional components related to cell wall remodeling in EVs. Furthermore, functional and uptake analysis confirmed the cell wall remodeling properties of yeast EVs. This study confirms that EVs can mediate cell wall remodeling under stressful conditions.