Background: Echinochloa esculenta is the Indian Barnyard millet which is a grass type of species. This study focused on the evaluation of the antibacterial activity of Echinochloa esculenta-mediated zinc oxide nanoparticles (ZnONPs) against urinary tract infection (UTI) microbes, such as Escherichia coli (E. coli), Enterobacter aerogenes, Pseudomonas aeruginosa, Staphylococcus aureus, and Proteus vulgaris. Methods: The millet Echinochloa esculenta-mediated ZnONPs are synthesized using a green method. Synthesized ZnONPs are characterized by ultraviolet (UV), Fourier transform-infrared resonance spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive X-ray analysis (EDX) to determine the functional groups, crystalline size, shape, and elemental composition of the synthesized NPs. Results: The UV and FTIR results of Echinochloa esculenta-mediated ZnONPs preliminarily confirmed the formation of ZnONPs. The XRD and SEM results also confirmed that the synthesized ZnONPs are well-crystalline in nature, and the average size was observed to be 23.38 nm with a spherical shape. Echinochloa esculenta-mediated ZnONPs contained 73.33% of zinc and 26.77% of oxygen, which was examined by EDX. Synthesized ZnONPs were tested against the five UTI pathogens, and they exhibited a greater zone of inhibitions than zinc acetate and plant extract. Especially against S. aureus, Echinochloa esculenta-mediated ZnONPs performed well with an inhibitory effect. Conclusions: Therefore, this study had the potential value of developing new eco-friendly, low-cost, and less toxic nanomaterials that can be used as a bio-control for diseases.