In this work, we fabricated ZnO and ZnO/CuO nanocomposites using Mentha longifolia leaf extract as a natural, non-toxic, and efficient stabilizer. Anti-bacterial activities of the prepared samples against two pathogenic bacteria, Escherichia coli (Gram-negative), and Staphylococcus aureus (Gram-positive) were investigated. The properties of the as-prepared samples were characterized by XRD, EDX, SEM, TEM, TGA, FT-IR, UV–Vis DRS, and BET instruments. The XRD analysis indicated that the size of crystallites was decreased for the ZnO powder prepared in the presence of the leaf extract. The SEM images showed that the samples consist of spherical shaped well-distributed particles. In addition, the presence of biomolecules from the leaf extract was revealed by EDX, TGA, FT-IR, and UV–Vis DRS analyses, which are important in biosynthesis process. The highest anti-bacterial activity belonged to the ZnO/CuO (10%) nanocomposite and the other compounds, including ZnO/CuO (5%), ZnO (ext), and ZnO (W) were in the next ranks, respectively. It was observed that the viability percentages against E. coli (10.16 ± 2.2) is higher than that of S. aureus (17.1 ± 0.87) in the presence of the ZnO/CuO (10%) nanocomposite. Ultimately, the mechanism for the action of the ZnO/CuO (10%) nanocomposite was explored through the SEM images, which involved the disruption of the bacterial membranes.