Recent trends and developments in nanotechnology and nanoscience have attracted wide interest in metallic nanoparticles owing to their excellent drug delivery and antimicrobial potential. This study focused on the green synthesis of copper oxide nanoparticles (CuO Np) from the plant Vitex negundo, an aromatic shrub mostly found in tropical areas. This plant has mainly utilized for its antioxidant activity and abundant flavonoids, terpenoids, and other secondary metabolites. In this study, CuONps were synthesized by the green synthesis process. They were characterized by using a UV-visible spectrometer, particle distribution analysis by DLS, FT-IR spectroscopy, XRD, and SEM & EDX. The CuONps were observed as crystalline structure, absorbing UV reading was 378 nm wavelength, spherical as seen in SEM images, and size was approximately 100 nm. Antimicrobial activity was assessed by agar well diffusion assay, a comparatively efficient inhibition zone of 8 mm was observed for CuO Np and 6 mm inhibition zones streptomycin against B. subtilis. Subsequently, anticancer activity was analyzed using a cell line assay, and the synthesized nanoparticles showed significant anticancer activity against HepG2 cell lines rather than HeLa cell lines. The half-maximal inhibitory concentration IC50 of CuO Np was observed as 49 & mu;g/ml and 82 & mu;g/ml, respectively, in HepG2 and HeLa cells. Further, more investigations are needed to unravel the full potential of these nanoparticles.