The development of effective treatments for dental pathogens and oral cancer remains a significant challenge. Copper oxide nanoparticles (CuO NPs) are recognized for their strong antimicrobial properties, attributed to the synthesis of oxygen-dependent radicals. α-Mangostin (MG), a natural xanthone from mangosteen fruit, is well-known for its antioxidant, antimicrobial, and anticancer potential. The combination of CuO NPs with MG would offer a synergistic approach to enhance therapeutic efficacy. CuO-MG NPs were synthesized and characterized for their size, morphology, and surface properties. The antimicrobial efficacy of these nanoparticles was tested against oral pathogens, including Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans, and Candida albicans. Antioxidant activity was assessed using superoxide anion and hydroxyl radical anion. The anticancer potential was evaluated by examining apoptosis induction in oral cancer cell lines, focusing on the expression of key apoptotic markers such as Caspase-3, Caspase-8, and FasL. Molecular docking simulations were performed to understand the interaction between MG and biofilm receptors. The CuO-MG NPs evidenced significant antimicrobial efficacy against all tested oral pathogens, with enhanced efficacy attributed to the combined effects of CuO-induced oxidative stress and the antimicrobial properties of MG. Antioxidant assays demonstrated a dose-dependent increase in radical scavenging activity. In oral cancer cells, CuO-MG NPs significantly reduced cell viability and induced apoptosis, as evidenced by the up-regulation of Caspase-3, Caspase-8, and FasL. Molecular docking studies revealed strong binding affinities of MG to key biofilm receptors, disrupting pathogen adhesion and biofilm formation. The combination of CuO NPs and MG offers a powerful and multifaceted therapeutic approach to oral healthcare. CuO-MG NPs demonstrate synergistic antimicrobial, antioxidant, and anticancer properties, offering a potential approach for the management of oral infections and oral cancer. Further preclinical and clinical studies are recommended to ensure their safety and stability in medical applications.