The button mushroom, Agaricus bisporus, is one of the major economical crops cultivated in Korea. This mushroom showed the 5th production to 11,493 M/T in 2014. Several fungus are known as the causal agents of diseases of the cultivated button mushroom (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus). Cladobotryum mycophilum is the causal agent of cobweb disease of commercial mushrooms. Early symptoms were noticed as round, fleshy, yellowish brown lesions on mushroom caps. Late symptoms progressed when the parasitic fungus formed white cobweb circular colonies on dead or damaged pinheads, spread on the surface of the casing, and covered entirely fruiting bodies. A Gram-positive bacterium was isolated from mushroom media that markedly showed the antagonistic activity against Cladobotryum mycophilum, the most destructive pathogen of cultivated mushrooms. The HC57 strain was selected as antagonistic bacterium by inhibition zone method and it was identified as Bacillus subtilis by the cultural, morphological and physiological characteristics, and analysis of the 16S rDNA. The isolated bacterium is saprophytic but not parasitic nor pathogenic to cultivation mushroom. The isolated bacterium for Cladobotryum mycophilum cell, was sufficient for inhibition in vitro. Inoculation of the isolated bacterium prevents the development of bacterial disease in Cladobotryum mycophilum. Control efficacy of browning disease of strain HC57 treatment was 71% on Agaricus bisporus. The optimal culture medium for the antagonistic bacteria growth was determined as follows: 1.5% Xylose, 2% Soytone, 1% NH4H2PO4, 7 mmol CaCl2, and 0.5% Histidine at pH 6.0 at 25℃. The suppressive bacterium may be useful in future for the development of biocontrol system and the construction of genetically modified edible fungi resistant to the disease caused by Cladobotryum mycophilum.