The psychrotolerant yeast Mrakia frigida 2E00797 isolated from sea sediment in Antarctica was found to be able to produce killer toxin against the pathogenic yeast (Metschnikowia bicuspidata WCY) in crab. When the psychrotolerant yeast was grown in the medium with pH 4.5 and 3.0% (wt/vol) NaCl and at 15°C, it could produce the highest amount of killer toxin against the pathogenic yeast M. bicuspidata WCY. The crude killer toxin activity against the pathogenic yeast M. bicuspidata WCY was the highest when it grew at 15°C in the assay medium with 3.0% (wt/vol) NaCl and pH 4.5. At temperatures higher than 25°C, the killing activity produced by M. frigida 2E00797 was completely lost and after the crude killer toxin was pre-incubated at temperatures higher than 40°C for 4 h, the killing activity was also completely lost. The killer toxin produced by M. frigida 2E00797 could kill only M. bicuspidata WCY, Candida tropicalis and Candida albicans among all the fungal species and bacterial species tested in this study.