Copper nanoparticles (CuNPs) have gained significant attention due to their wide range of applications, particularly in antibacterial treatments. In this study, we synthesized CuNPs using an eco-friendly mycosynthesis approach with Pleurotus ostreatus (oyster mushroom) extract and employed an autoclave-assisted method to enhance the synthesis process. This green synthesis method presents distinct advantages, such as reduced toxicity, cost-effectiveness, and environmental sustainability. The copper nanoparticles were characterized using UV-Vis spectroscopy, field emission scanning electron microscopy (FESEM), and Fourier-transform infrared spectroscopy (FTIR). UV-Vis spectroscopy confirmed the formation of CuNPs with a peak at 374 nm. FESEM revealed irregular shapes and a wide size distribution, ranging from 41.56 nm to 131.9 nm. FTIR analysis demonstrated the presence of functional groups acting as capping agents, stabilizing the nanoparticles. The root elongation test and inter simple sequence repeat (ISSR)-PCR showed that the CuNPs exhibited no toxic effects on rice root growth. The findings suggest that autoclave-assisted mycosynthesis of CuNPs using P. ostreatus offers a sustainable and efficient route for producing CuNPs with potential applications in antimicrobial treatments and agricultural industry.