AbstractThe potential for using cotton seed hulls (CSHs) and walnut shells (WSs) as new, essential substances for substrate preparation in the cultivation of Pleurotus ostreatus was studied. Substrates prepared with oak sawdust alone (OS) and with mixtures of OS and CSHs and WSs in different ratios were compared, and their effects on the earliness, total time, yield, and biological efficiency (BE) were determined. The nitrogen (N) content of the substrates prepared using CSHs and WSs alone was high, so the C:N ratio of the substrates diminished as the proportions of CSHs and WSs in the mixtures were increased. The highest yields were obtained from substrates containing the maximum amount of N. The highest yield and highest biological efficiency were obtained for a mixture of 25OS:75CSHs, indicating that the yield in the substrates increased as the amount of CSHs in the mixtures increased. The morphological characteristics were influenced by the various substrates and their various ratios. The properties of mushroom cultivation in bags were related to the nitrogen content, as indicated by the C:N ratios. The results indicated that CSHs and WSs could be used as new, essential substances in the preparation of substrates for the cultivation of Pleurotus ostreatus.