This study was conducted to reuse spent mushroom substrates (SMS) of Pleurotus ostreatus and improve their nitrogen content by bacterial treatment. Two kinds of bacteria were used to investigate the increase in total nitrogen (T-N) content. Bacillus sp. (GM20-4) was isolated from SMS of oyster mushroom, and Rhodobacter sphaeroides (RS) was obtained from Gwangju Si Agricultural Technology Center. SMS samples were collected from three oyster mushroom cultivation farms located in Gyeonggi-do province, Korea. When dried SMS was inoculated with 30% culture broth of GM20-4 and RS and incubated at room temperature (25±2oC) for 5 days, T-N content increased. To investigate the T-N content of other SMS, three dried SMS samples (A, B, and C) were treated by the same method using GM20-4 and RS. As a result, the T-N content of sample B was 20% higher than that of the control, whereas the T-N content of samples A and C increased to 17% and 12%, respectively. The change in T-N content by bacterial treatment of wet SMS was slightly higher than that of the control. The changes in amino acid content were also found to be higher than those in the control in all SMS samples by GM20-4 and RS treatment. Aspartic acid and glutamic acid contents were the highest among all amino acid compositions. Especially, the aspartic and glutamic acid contents of sample B increased by 2.9 folds higher than the control.