The sustainable management of winery residues could represent a cornerstone for promoting environmental and economic sustainability from a circular economy perspective. In this context, our study aimed to evaluate Vitis vinifera L. ‘Nero di Troia’ cultivar grape pomace as a valuable waste product for the cultivation of Pleurotus mushroom, in comparison with traditional wheat straw-based cultivation. Mushroom extracts were prepared through the eco-friendly microwave-assisted extraction technique, using green solvents with different polarity degrees. Total protein content, total polyphenol content, and antioxidant activity (FRAP and DPPH assays) were assessed for the water and EtOH hydrophilic extracts. Grape pomace often gave higher values than wheat straw, especially for the P. eryngii var. eryngii water extract protein content, which was 3.5-fold higher (0.68 ± 0.14 mg BSA/mL and 0.192 ± 0.025 mg BSA/mL, respectively). The ethyl acetate extracts of both mushroom species gave biologically relevant results in terms of inhibiting activity against acetylcholinesterase, an enzyme involved in the pathogenesis of Alzheimer’s disease (50% inhibitory activity at concentrations ≤ 1.5 mg/mL), thus paving the way for more in-depth investigation. The extract’s metabolic profile was investigated through GC-MS analysis. The results show that incorporating grape pomace into mushroom production represents a concrete step toward more sustainable biotechnological processes. © 2025 by the authors.

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