The antagonistic effect exerted by Saccharomyces cerevisiae against other microbial species during wine fermentations was recently ascribed to its capacity to secrete antimicrobial peptides (AMPs). The main goal of the present work was to purify, identify and characterize those AMPs. Firstly, the AMPs were purified by means of chromatographic techniques (sizee-xclusion and ion-exchange) and then characterized regarding their amino acid sequence, codifying genes and antimicrobial/biochemical properties. Analysis of the purified AMPs by mass spectrometry revealed that the natural biocide is mainly composed by two peptides (AMP1 and AMP2/3) derived from the isoenzymes of the glycolytic protein glyceraldehyde 3- phosphate dehydrogenase (GAPDH). The spectrum of action of the naturally-excreted AMPs, which we named saccharomycin, is wide and includes several wine-related non-Saccharomyces yeasts, such as Hanseniaspora guilliermondii, Torulaspora delbrueckii, Kluyveromyces marxianus, Lachancea thermotolerans and Dekkera bruxellensis, as well as bacteria such as Oenococcus oeni. The antimicrobial effect of saccharomycin is significantly higher than that of synthetic analogues (AMP1 and AMP2/3) and depends on their complementary action and relative proportion. The mode of action of the AMPs was evaluated against sensitive yeast cells. The AMPs induce cell membrane permeabilization, loss of pH homeostasis and increase/decrease of H+-influx/-efflux. They also induce cell molecular markers typical of death by apoptosis in H. guilliermondii. Our work also revealed the accumulation of these GAPDHderived peptides on the surface of stationary-grown (48 h) cells of S. cerevisiae, which induce death of non-Saccharomyces yeasts (H. guilliermondii and L. thermotolerans) by direct cell-cell contact. Finally, S. cerevisiae strains over-expressing these AMPs prevented growth of D. bruxellensis in co-fermentations, decreasing the levels of sulphur dioxide needed to control wine spoilage. Thus, the potential of these AMPs to be used as biopreservative in wine seems promising.