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Zinc oxide nanoparticles (ZnO NPs) are versatile and promising, with diverse applications in environmental remediation, nanomedicine, cancer treatment, and drug delivery. In this study, ZnO NPs were synthesized utilizing extracts derived fromAcacia catechu, Artemisia vulgaris, andCynodon dactylon. The synthesized ZnO NPs showed an Ultraviolet–visible spectrum at 370 nm, and X-ray diffraction analysis indicated the hexagonal wurtzite framework with the average crystallite size of 15.07 nm, 16.98 nm, and 18.97 nm for nanoparticles synthesized utilizingA. catechu, A. vulgaris,andC. dactylonrespectively. Scanning electron microscopy (SEM) demonstrated spherical surface morphology with average diameters of 18.5 nm, 17.82 nm, and 17.83 nm for ZnO NPs prepared fromA. catechu, A. vulgaris, andC. dactylon,respectively. Furthermore, ZnO NPs tested againstStaphylococcus aureus, Kocuria rhizophila, Klebsiella pneumonia,andShigella sonneidemonstrated a zone of inhibition of 8 to 14 mm. The cell viability and cytotoxicity effects of ZnO NPs were studied on NIH-3T3 mouse fibroblast cells treated with different concentrations (5 μg/mL, 10 μg/mL, and 50 μg/mL). The results showed biocompatibility of all samples, except with higher doses causing cell death. In conclusion, the ZnO NPs synthesized through plant-mediated technique showed promise for potential utilization in various biomedical applications in the future.