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Bottom mushroom (Agaricus bisporus) is an edible basidiomycete mushroom in Europe and North America. The wheat straw, wheat brain and chicken manure were used for bottom mushroom cultivation. Bottom mushroom substrates for cultivation was collected from bottom mushroom farm in Buyeo. About 16 bacteria were isolated from fresh mushroom substrates on TSA medium. Among of the isolates, isolate 3HS12 possessed high cellulase and mannase activity was selected as a hydrolase-producing bacteria. The isolate 3HS12 was identified as members of the Bacillus subtilis by biochemical characteristics using Bacillus ID kit and VITEK 2 system. Comparative 16S rDNA gene sequence analysis showed that isolate 3HS12 formed a distinct phylogenetic tree within the genus Bacillus and was most closely related to Bacillus subtilis with 16S rDNA gene sequence similarity of 98.8%. On the basis of its physiological properties, biochemical characteristics and phylogenetic distinctiveness, the 3HS12 isolate was assigned to the Bacillus subtilis. The cellulase and mannase activity of Bacillus subtilis 3HS12 was increased according to the bacterial density at 40°C.