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In this study, Ziziphus spina christi leaves was used to synthesize a trimetallic CuO/Ag/ZnO nanocomposite by a simple and green method. Many characterizations e.g. FTIR, UV-vis DRS, SEM-EDX, TEM, XRD, zeta-size analysis, and DLS, were used to confirm green-synthesized trimetallic CuO/Ag/ZnO nanocomposite. The green, synthesized trimetallic CuO/Ag/ZnO nanocomposite exhibited a spherical dot-like structure, with an average particle size of around 7.11 ± 0.67 nm and a zeta potential of 21.5 mV. An extremely homogeneous distribution of signals, including O (79.25%), Cu (13.78%), Zn (4.42%), and Ag (2.55%), is evident on the surface of green-synthetic nanocomposite, according to EDX data. To the best of our knowledge, this is the first study to effectively use an industrially produced green trimetallic CuO/Ag/ZnO nanocomposite as a potent antimicrobial agent by employing different statistically experimental designs. The highest yield of green synthetic trimetallic CuO/Ag/ZnO nanocomposite was (1.65 mg/mL), which was enhanced by 1.85 and 5.7 times; respectively, by using the Taguchi approach in comparison to the Plackett-Burman strategy and basal condition. A variety of assays techniques were utilized to evaluate the antimicrobial capabilities of the green-synthesized trimetallic CuO/Ag/ZnO nanocomposite at a 200 µg/mL concentration against multidrug-resistant human pathogens. After a 36-h period, the tested 200 µg/mL of the green-synthetic trimetallic CuO/Ag/ZnO nanocomposite effectively reduced the planktonic viable counts of the studied bacteria, Escherichia coli and Staphylococcus aureus, which showed the highest percentage of biofilm reduction (98.06 ± 0.93 and 97.47 ± 0.65%; respectively)., (© 2024. The Author(s).)