The wide diffusion of organic cultivation of vineyards and the need to reduce the use of pesticides highlights the urgent need for alternative and sustainable methods of vine protection by pathogen molds. Biocontrol of molds by wine yeast species has been suggested as a promising tool to avoid or limit the chemical fungicides. The present study aims to evaluate and characterize the antifungal activity of several non-Saccharomyces yeast species isolated from different environmental and food sources against Botrytis cinerea and other spoilage fungi of grapes such as Aspergillus carbonarius, Penicillium expansum, Penicillium digitatum and Cladosporium. A preliminary evaluation of metabolites and volatile compounds with antimicrobial action was carried out. Several yeast strains mainly belonging to Brettanomyces bruxellensis and Metschnikowia pulcherrima but also to other species showed a wide antifungal activity. A selected strain of M. pulcherrima was then evaluated in the field. Fifteen days before harvest, the strain was sprayed on both red and white grape batches. At vintage time (after 15 days) the batches were evaluated for biocontrol and grape colonization. Results showed significant colonization of the M. pulcherrima strain and valuable mold control. These findings highlight the potential of yeast-based biocontrol strategies as sustainable and effective tools for vineyard management.