The global crisis of antibiotic resistance requires the creation of alternative therapeutics. Metal oxide nanoparticles are considered a novel class of biologically active materials. This study investigated the antimicrobial and anticancer effects of copper oxide nanoparticles (CuO-NPs) and their doped form with silver, which were synthesized using okra fruit extract. The X-ray diffraction (XRD) spectrum indicated that all NPs had a monoclinic structure, and the crystal size of the NPs increased with doping. The spectrum obtained from ultraviolet-visible diffuse reflectance spectroscopy (UV-Vis/DRS) analysis of NPs exhibited an absorption band at a wavelength of 365 nm. The formation of CuO-NPs, with an average size of 57 nm, was confirmed by the field emission scanning electron microscopy (FESEM) analysis. Due to doping with Ag, the size of the NPs increased. The morphology of the pure CuO-NPs, as observed in the FESEM micrographs, appeared to be spherical with good dispersion. The CuO-NPs doped with Ag displayed rectangular and rod-like morphologies. The CuO-NPs doped with Ag exhibited the highest level of inhibition against both bacterial and fungal isolates. The P. aeruginosa displayed the highest sensitivity with an inhibition zone of 23 mm among bacterial isolates. Among the fungal isolates, Candida and Rhizopus exhibited inhibition at lower concentrations and demonstrated heightened sensitivity compared to the Aspergillus species. The study investigated cytotoxicity using the MTT method on HeLa cancer cells. It showed that increasing NPs concentration decreased cell viability, indicating dose-dependent cytotoxicity.