This study investigated the incorporation of Amphipterygium adstringens ethanolic extract into the synthesis of copper oxide nanoparticles (CuO+Aa) and evaluates their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Acinetobacter baumannii (CRAB). To assess the impact of the extract, chemically synthesized CuO nanoparticles (CuO-NPs) and the extract alone were also tested. Both CuO-NPs and A. adstringens are known for their antimicrobial properties.CuO+Aa nanoparticles were synthesized using A. adstringens extract and characterized through Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), Zeta Potential, Thermogravimetric Analysis (TGA), and X-ray Diffraction (XRD), and compared to CuO-NPs. The influence of the extract was analyzed using Inductively Coupled Plasma Mass Spectroscopy (ICP-MS), UV-Vis, FTIR, Raman, and Nuclear Magnetic Resonance Spectroscopy (NMR). Antibacterial effects were tested using the microdrop technique and biofilm inhibition. Bacterial structural changes were observed via Scanning Electron Microscopy (SEM), and cytotoxicity was measured through hemolysis assays.CuO+Aa nanoparticles were smaller (3.46 nm) than CuO-NPs (5.32 nm). TGA indicated improved thermal degradation in CuO+Aa, suggesting incorporation of organic compounds. XRD revealed a shift from CuO to a mixed CuO-Cu2O phase (75.15%-24.84%) in CuO+Aa due to the functional groups present in the extract. Antibacterial assays showed that CuO+Aa inhibited MRSA and CRAB by 77% and 49%, respectively, using only 17.5 ppm of copper oxides-significantly lower than CuO-NPs, which required 150 ppm to achieve 96% and 78% inhibition. SEM revealed bacterial surface damage, including roughness, perforations, and cell wall collapse. All treatments showed low cytotoxicity (<2% hemolysis). Biofilm formation increased by 180% in MRSA and 131% in CRAB.A. adstringens ethanolic extract enhances CuO nanoparticle synthesis, reducing size and maintaining strong antibacterial activity with low toxicity. CuO+Aa represents a promising candidate for future biomedical applications against resistant pathogens.