The increasing prevalence of cancer necessitates the development of novel and effective therapeutic agents. This study evaluates the anticancer potential of biosynthesized copper oxide nanoparticles (CuO NPs) using extract against human colon and breast cancer cells. The CuO NPs were characterized using various techniques to confirm their structure, size, morphology, and functional groups. The average size of CuO NPs synthesized was 20-60 nm, with spherical shape. The cytotoxic effects of these CuO NPs reveal a dose-dependent reduction in cell viability with 50% inhibitory concentration (IC) at 58.53 ± 0.13 and 53.95 ± 1.1 μg/mL, respectively. Further investigation into the mechanism of action was conducted using flow cytometry and apoptosis assays, which indicated that CuO NPs induced cell cycle arrest and apoptosis in cancer cells. Reactive oxygen species (ROS) generation, caspase activity assay, and comet assay were also performed to elucidate the underlying pathways, suggesting that oxidative stress and DNA damage play pivotal roles in the cytotoxicity observed. Overall, our findings demonstrate that biosynthesized CuO NPs exhibit notable anticancer activity against colon and breast cancer cells, with moderate selectivity over normal cells, highlighting their potential as a therapeutic agent due to their biocompatibility. However, further studies are required to validate their selectivity and safety profile.