Aims In the present study, it was investigated the antagonistic behaviour of Metschnikowia pulcherrima, as biocontrol agent, against the main wine yeast species involved in the winemaking process. Methods and Results Seven strains of M.pulcherrima were evaluated for the antimicrobial activity against 114 yeast strains belonging to Pichia, Candida, Hanseniaspora, Kluyveromyces, Saccharomycodes, Torulaspora, Brettanomyces and Saccharomyces genera. Results showed both different inter-generic and intra-generic responses to the antimicrobial action of M.pulcherrima strains. Interestingly, the antimicrobial activity of M.pulcherrima did not have any influence on the growth of Saccharomyces cerevisiae. Instead, M.pulcherrima displayed a broad and effective antimicrobial action on undesired wild spoilage yeasts, such as Brettanomyces/Dekkera, Hanseniaspora and Pichia genera. Fermentation trials carried out in synthetic grape must confirmed the antimicrobial activity of M.pulcherrima, determining the early death of the non-Saccharomyces co-inoculated cultures. Conclusions The antimicrobial activity of M.pulcherrima does not seem due to proteinaceous compounds such as killer phenomenon, but to the pulcherriminic acid (the precursor of pulcherrimin pigment) that depletes iron present in the medium, making it not available to the other yeasts. Significance and Impact of the Study These data agree with and further support the potential use of selected M.pulcherrima strains in controlled multistarter fermentations with S.cerevisiae starter cultures.