The global emergence and tenacity of multidrug-resistant microbes have raised new challenges for the management of diseases associated with infections. Metal-based nanoparticles (NPs) have recently received special attention as a prospective alternate for existing chemical antibiotics because of their extensive antibacterial potency and low toxicity. Herein, copper nanoparticles (CuONPs) were prepared by using an aqueous extract of aerial parts Rhus microphylla (RM) aerial parts. The obtained RM-CuONPs were characterized and evaluated for antimicrobial and antibiofilm potential against various human pathogens. The formed RM-CuONPs were well dispersed with a uniform spherical shape and an average size of 32.45 nm. Numerous functional moieties found in the FTIR spectra confirmed that the phytocomponents of the RM-extract were in charge of the synthesis synthesis, capping, and stabilization of RM-CuONPs. The biogenic RM-CuONPs demonstrated superior antibacterial effectiveness towards Staphylococcus epidermidis and Enterobacter cloacae with a minimum inhibitory concentration (MIC) value of 48.5 µg·mL−1. Remarkable antifungal activity of RM-CuONPs was noted against C. tropicalis (MIC = 97 µg·mL−1). Also, the biosynthesized RM-CuONPs demonstrated notable potential in reducing biofilm formation in a dose-dependent manner. These inferences offer an insight into the plausible for utilizing plant extracts for the biosynthesis of CuONPs with enhanced biological activity and could offer promising effective substitutes to traditional antimicrobials for the treatment of biofilms and drug-resistant bacteria.