Biosynthesis of metal nanoparticles presents a promising approach for their efficient and environmentally friendly production. In this study, CuO nanoparticles were successfully synthesized by using Rumex nepalensis Spreng. as a bio-reducing agent. The spectroscopic analysis confirmed the crystalline monoclinic structure of the synthesized CuO NPs, with particle sizes ranging from 21 to 97 nm. These biosynthesized CuO NPs exhibited notable antimicrobial activity against diverse microorganisms, suggesting their potential for antimicrobial applications. Moreover, the CuO NPs displayed significant antioxidant activity, demonstrated by their effective scavenging of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radicals. This study highlights the straightforward, cost-effective, non-toxic, and robust nature of CuO NPs synthesis using Rumex nepalensis Spreng., offering insights into their potential applications in antimicrobial and antioxidant fields.