The objective of this study was to achieve biological control of green mold disease in Pyogo mushrooms using antagonistic microorganisms. Bacillus subtilis BSM320 cells inhibited mycelial growth by 48–60% against three Trichodermaisolates including T. hazianumisolated from the substrates of Lentinula edodes, showing their antifungal activity.The bacteria were cultured to a high density of 4.2 x 109±113.7 cfu/mlin aqueous extract of composted spent mushroom substrates of L. edodes containing 1% glucose and showed a higher growth rate than that observed when using the commercial medium, Luria-Bertani broth. The bacterial culture showed a 75% protective effect without damaging the mushroom fruiting bodies. These results suggest that B. subtilis BSM320culture is suitable for biological control of green mold disease during mushroom cultivation.