This study focuses on the biological examination of copper oxide nanoparticles (CuO NPs) synthesized using Catharanthus roseus leaf extract, which served both as reducing and stabilizing agents. The formation of these biogenic CuO NPs was analyzed through UV-visible, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The UV-visible spectral analysis revealed a peak at 273 nm, while FTIR provided insight into the functional groups associated with the NPs. Antibacterial testing showed inhibition zones (ZOI) and Minimum Inhibitory Concentration (MIC) against eight pathogenic bacterial strains, both positive and negative. Antifungal testing exhibited ZOI against four pathogenic fungal organisms. Furthermore, the nanoparticles displayed antioxidant potential, with an IC50 value of 58.55 µg/ml, indicating the scavenging ability. The in-vitro assessment of anticancer activity was carried out against the HT29 cell line, revealing an IC50 value of 37.99 µg/ml.