This research investigated the physicochemical properties and biological activities of green-synthesized copper oxide nanoparticles (CuO NPs) via Moringa peregrina extract, graphene oxide (GO), and their composite (CuO–GO). SEM revealed the morphology and structure, indicating polygonal CuO NPs, thin wrinkled sheets of GO, and a combination of CuO NPs and GO in the nanocomposite. EDS confirmed the elemental composition and distribution. XRD analysis confirmed the crystalline monoclinic structure of CuO NPs and GO, as well as their composite, CuO–GO, with characteristic peaks. DLS analysis exhibited distinct size distributions, with CuO NPs showing the narrowest range. BET surface area analysis revealed mesoporous structures for all materials, with the nanocomposite showing enhanced surface area and pore volume. Anticancer assays on MCF-7 and normal NIH/3T3 cells demonstrated CuO–GO’s superior cytotoxicity against cancer cells, with minimal effects on normal cells, suggesting selective cytotoxicity. Moreover, antibacterial assays against Pseudomonas aeruginosa and Staphylococcus aureus indicated CuO–GO’s potent inhibitory activity. The composite’s synergistic effects were evidenced by its lower minimum inhibitory concentration (MIC) compared to individual components. In conclusion, this study elucidated the promising biomedical applications of CuO NPs, GO, and their nanocomposite, particularly in cancer treatment and antibacterial therapies, showcasing their potential as multifunctional nanomaterials.