This study was conducted to investigation species of microbes collected form SMS(spent mushroom substrate) of oyster mushroom. SMS samples were collected from two farms and mushroom research Institute in Gyeonggi-Do. 52 microbial isolates form SMS of oyster mushroom were collected. 24 of these isolates identified Bacillus sp. By using 16s rDNA. Among the isolates, Bacillus sp. was the most abundant with 46% of total isolates, followed by Lactococcus sp. with 20% and Saccharomycetales contained Galactomyces sp. with 19%. To define the optimum conditions for the cell mass of major isolates collected from SMS of oyster mushroom, we have investigated optimum culture condition such as pH, temperature, and incubation time. Optimum initial pH, temperature and incubation time of Bacillus sp. were determined as pH 6.0, 25°C and 48 hours, respectively. In the Lactococcus sp, optimum initial pH 6.0, optimum initial temperature 35°C and optimum growth period 48 hours. Optimum initial conditions of Saccharomycetales were investigated as pH 7.0, temperature 25°C and growth period 36 hours, respectively.