Bacterial resistance to conventional antibiotics has created an urgent need to develop enhanced alternatives. Nanocomposites combined with promising antibacterial nanomaterials can show improved antimicrobial activity compared to that of their components. In this work, green synthesized CuO nanoparticles (NPs) supported on an anionic clay with a hydrotalcite-like structure such as Zn−Al layered double hydroxide (LDH) nanocomposite were investigated as antimicrobial agents. This nanocomposite was synthesized using Micromonospora sp. ISP-2 27 cell-free supernatant to form CuO NPs on the surface of previously synthesized LDH. The prepared samples were characterized using UV−Vis spectrophotometry, XRD, FTIR, Field emission scanning electron microscopy with EDX, zeta potential, and hydrodynamic particle size. UV−vis spectral analysis of the biosynthesized CuO NPs revealed a maximum peak at 300 nm, indicating their successful synthesis. The synthesized CuO NPs had a flower-like morphology with a size range of 43−78 nm, while the LDH support had a typical hexagonal layered structure. The zeta potentials of the CuO NPs, Zn−Al LDH, and CuO NPs/LDH nanocomposite were −21.4, 22.3, and 30.8 mV, respectively, while the average hydrodynamic sizes were 687, 735, and 528 nm, respectively. The antimicrobial activity of the produced samples was tested against several microbes. The results demonstrated that the nanocomposite displayed superior antimicrobial properties compared to those of its components. Among the microbes tested, Listeria monocytogenes ATCC 7644 was more sensitive (30 ± 0.34) to the biosynthesized nanocomposite than to CuO NPs (25 ± 0.05) and Zn−Al LDH (22 ± 0.011). In summary, the use of nanocomposites with superior antimicrobial properties has the potential to offer innovative solutions to the global challenge of antibiotic resistance by providing alternative treatments, reducing the reliance on traditional antibiotics, and contributing to the development of more effective and targeted therapeutic approaches.