| Summary: | The feasibility of utilising spent mushroom substrates (SMSs) as a growing medium component for <i>Pleurotus ostreatus</i> cultivation was investigated. <i>P. ostreatus</i> was cultivated on traditional wheat straw (control) and wheat straw substrate supplemented with SMSs from <i>Pholiota nameko</i> (N-SMS), <i>Hypsizygus marmoreus</i> (M-SMS), and <i>Hericium erinaceus</i> (E-SMS) in varying supplementation rates (10%, 20%, and 30%). The yield, biological efficiency (BE), dry matter, and protein content of <i>P. ostreatus</i> fruiting bodies grown on ten substrates were evaluated. Significant differences in yield, BE, protein content, and dry matter of <i>P. ostreatus</i> were found among the studied substrates. The highest yield was recorded in 20% E-SMS (254.33 g), 20% N-SMS (253.43 g), and 10% E-SMS (251.67 g). The biological efficiency ranged from 66.48% (30% M-SMS) to 72.67% (20% E-SMS) and followed a similar trend to yield. The highest protein content was recorded in 30% M-SMS (29.93 g∙100 g dry weight<sup>−1</sup>). The highest dry matter of <i>P. ostreatus</i> was noticed in 30% of M-SMS (23.74 g) and 10% of M-SMS (23.06 g). Therefore, the spent mushroom substrates of <i>Ph. nameko</i>, <i>H. marmoreus</i>, and <i>H. erinaceus</i> could be used as a potential, low-cost sustainable alternative (10–30%) and as a renewable component of traditional growing media for <i>P. ostreatus</i> cultivation.
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