Biosynthesis techniques for nanomaterials have advanced significantly, promoting eco-friendly synthesis chemistry as a sustainable alternative to conventional methods. This study presents a novel and environmentally friendly approach for synthesizing nanoparticulate ZnO, CuO, and ZnO/CuO nanocomposites using aqueous extracts of Pleurotus ostreatus spent substrate, is reported. The structural, optical, and morphological properties of the synthesized NPs were analysed. A hexagonal phase of ZnO NPs and a monoclinic phase of CuO NPs were obtained according to the X-ray diffraction analysis. A reduction in the peak intensity of these metal oxides was observed in the ZnO/CuO NPs due to reduced crystallinity. The absorption spectra, obtained from the UV-vis analysis, showed peaks at 354, 365, and 525 nm for the ZnO, CuO, and ZnO/CuO NPs, respectively. An anticancer assay of the NPs was conducted using human embryonic kidney (HEK 293) and cervical carcinoma (HeLa) cell lines, while a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was used for the antioxidant evaluation. The ZnO, CuO, and ZnO/CuO NPs showed higher antioxidant potency with IC