The aim of this study was to re-use spent mushroom substrate (SMS) with increased total nitrogen (T-N) and amino acid content and reduce the amount of cottonseed meal used as nutrient supplement in Pleurotus ostreatus cultivation. Bacteria used for improvement of the T-N content were GM20-4(Bacillus sp.) and Rhodobacter sphaeroides (RS). GM20-4 was isolated from the SMS of P. ostreatus and RS was obtained from Gwangjusi agricultural technology center. SMS in T1, T2, and T3 was reused as substrate after drying and the T-N content of dried SMS (D-SMS) was increased by 0.34% by treatment with the bacteria. T1 with 8% D-SMS and T2 with 18% D-SMS had higher rates of primordia formation compared with T3 and the control. The biological efficiency of the control and of treatment with 8%, 18%, and 26% D-SMS was 110%, 114%, 112%, and 79%, respectively. Considering the economic cost, yield, and biological efficiency, T2 with 18% D-SMS as the culture substrate for P. ostreatus was shown to be the most effective for cultivation.