Bacillus subtilis H27, an isolate from a commercial cheonggukjang product, inhibited Listeria monocytogenes ATCC 19111 and Bacillus cereus ATCC 14579. Antimicrobial activity of culture supernatant was completely destroyed by proteinase K treatment, indicating the proteinaceous nature of the inhibitory substance. The bacteriocin, Bac H27, was most stable at pH 7 and stable between pH 3-6 and 8-9 but inactive at pH 10 and above. The activity reduced to 50% after 15 min at $80^{circ}C$. Brain heart infusion (BHI) was the best medium for the Bac H27 activity (160 AU/mL). Bac H27 was purified by ammonium sulfate precipitation followed by Q-Sepharose and Sephadex G-75 column chromatographies. Its size was estimated to be 4.9 kDa by Tricine SDS-PAGE. Partially purified Bac H27 killed L. monocytogenes ATCC 19111 when added into L. monocytogenes culture. Bac H27 could be useful as a novel food grade preservative inhibiting B. cereus and L. monocytogenes, 2 important food pathogens.