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Pleurotus ostreatus, an edible mushroom widely consumed worldwide, generates a by-product known as spent mushroom substrate (SMS). This material has demonstrated biological activity against agricultural crop pathogens. In this study, we evaluated the nematocidal effectiveness of hydroalcoholic extracts (T5, T2, AT5, and AT2) derived from SMS of P. ostreatus against (J2) of the phytonematode Nacobbus aberrans and assessed their potential toxicity towards the non-target nematode Panagrellus redivivus. Among these extracts, AT5 exhibited the highest efficacy against N. aberrans and was the least toxic against P. redivivus. Liquid–liquid partitioning yielded the AQU fraction, which showed significant nematocidal activity against J2 (75.69% ± 8.99 mortality), comparable to chitosan. The GC-MS analysis revealed the presence of several compounds, including palmitic acid, linoleic acid, and 2,4-Di-tert-butylphenol. These findings are consistent with studies confirming the antagonistic effectiveness of these compounds against phytonematodes. Additionally, all extracts exhibited toxicity against P. redivivus, with T2 being the most toxic. Our findings demonstrate that while the AT5 extract displays antagonistic effectiveness against both N. aberrans and P. redivivus, it was the least toxic among the extracts tested. Thus, SMS of P. ostreatus holds potential as a source of nematocidal compounds, which could offer significant benefits for agricultural pest control. Inglés