Probiotic yeast strains viz. Yarrowia lipolytica VIT-MN01, Kluyveromyces lactis VIT-MN02, Lipomyces starkeyi VIT-MN03, Saccharomycopsis fibuligera VIT-MN04 and Brettanomyces custersianus VIT-MN05 were investigated as producer of killer toxin. Among the five yeasts, three strains showed more killer toxin production. The maximum killer activity (12 aU/mg) against sensitive strain Saccharomyces cerevisiae was obtained at pH-3 with 0.5% NaCl at 25°C by using response surface methodology. The purified killer toxin K2 and K3 showed the highest killer activity against foodborne pathogens viz. Bacillus cereus, E. coli, Pseudomonas spp., Klebsiella spp. and Candida tropicalis. The purity of the killer toxin was confirmed by HPLC analysis. The molecular mass of killer toxin was 22 kDa (K2), 18 kDa (K3) and 14 kDa (K4). The results of the present study suggest that killer toxins produced by probiotic yeast strains can be used as an antimicrobial agent to control the microbial contamination in the food industry.