Agro-industrial by-products are potential environmental pollutants. Achieving sustainability leads us to think for handling them in an environmentally friendly way. The present framework was designed to investigate the potential of subsequent utilization of mushroom spent substrate (MSS) of oyster mushroom (Pleurotus ostreatus var. columbinus) cultivation. Two consecutive trials (2018 and 2019) were conducted at Mushroom Laboratory, Sohag University, Egypt. Fresh rice straw (RS) was used along with mushroom spent substrate (MSS) as control treatments and compared with another three different mixtures of RS:MSS as follows: 1:2, 1:1, and 2:1. The agronomic parameters of the new growing cycle of oyster mushroom, as well as the chemical compounds of substrates were investigated. Data revealed that pure (RS) substrate gave the greatest fruiting bodies yield and number/500g substrate, average fruiting bodies weight, and both of the cap diameter and stem length. In contrast, sole (MSS) gave the least values in both trials. Employing RS and MSS
substrate mixture at 2:1 ratio exceeded the other two formulations concerning the above-mentioned parameters. Relative to RS, the analyses of substrates nutritive compounds in MSS showed a significantly higher percentage of N, P, K, total carbohydrates and total ash after cultivation , but had a lower percent in total carbon, hemicellulose, cellulose, and lignin. In conclusion, SMS alone can›t be considered a potent substrate for further mushroom production. However, it may be used as a filler material in combination with RS but at low ratio.