Yeasts can produce toxins in protein or glycoprotein structures that can act as an inhibitor on some bacteria and yeast species. The effects of those toxins on the growth of pathogenic and food spoilage microorganisms are subject to various studies. Metschnikowia pulcherrima was determined to be a killer toxin-producing yeast that was tested against three selected microorganisms, namely Escherichia coli Type-I, Micrococcus luteus and Candida albicans. The killer toxin only showed inhibitory activity against M. luteus. Different pH (5-6-7-8), temperature (20-25-30-35 degrees C) and carbon source (glucose-glycerol-ethanol-acetate) combinations were applied to stimulate the growth and toxin production of the killer yeast. The greatest increase among the different combinations was obtained at 20 degrees C and pH 7 when glycerol was used as the main carbon source. It was then also tested against other pathogen indicators or pathogens under these conditions. The killer toxin was partially purified by ethanol precipitation and showed inhibitory activity against M. luteus (36 mm). According to the protein profile obtained by SDS-PAGE, the molecular weight of the inhibitor toxin was measured about 7.4 kDa. The molecular weight with amino acid sequence of the killer toxin was 10.3 kDa and determined by MALDI-TOF mass spectrometry.