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Zinc oxide nanoparticles (ZnO NPs) have acquired large attention against microbial activity because of their distinctive properties associated with their size and shape. In current research, ZnO NPs produced by the Sol-gel method at room temperature were applied to evaluate its antibacterial activity against pathogenic bacterial strains of Staphylococcus aureus and Escherichia coli obtained from Al-Sadr Hospital, Maysan, Iraq. The prepared nanoparticles were investigated by dynamic light scattering (DLS), X-ray diffraction (XRD), and scanning electron microscopy (SEM). Antibacterial activity was tested by using the agar diffusion method, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) after 24 h of exposure to prepared ZnO NPs. The size distribution, characterization, and morphology of nanoparticles showed around 100 nm as observed by DLS, and the formed nanoparticles were spherically confirmed by SEM. Additionally, results of the disc diffusion test showed significant inhibition of growth of both bacterial strains by ZnO NPs. In conclusion, ZnO NPs created by the sol-gel method had an excellent antibacterial effect, that could be used to produce ZnO NPs for biomedical applications. [ABSTRACT FROM AUTHOR]