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Here we report the biosynthesis of zinc oxide nanoparticles (ZnO-NPs) using aqueous extract of medicinally active Mentha mozaffarianii for biomedical applications. ZnO-NPs were characterized by X-ray diffraction (XRD), Field Emission Scanning Electron Microscope (FE-SEM), Energy-dispersive X-ray (EDX) spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy. The sharp and narrow diffraction peaks in the whole spectrum revealed that the ZnO-NPs are nanosized and well crystalized. FE-SEM analysis exhibited the spherical shape of the biosynthesized ZnO-NPs (mean diameter of about 20–29 nm). EDX analysis confirmed the formation of highly pure ZnO-NPs. ZnO-NPs exerted distinct effects on cancer cell lines while posing no impact on normal fibroblast cells. ZnO-NPs were primarily effective against HeLa (IC50: 50.1 μg/ml) cells, followed by MDA-MD231 (IC50: 54.9 μg/ml), and LS180 (63.4 μg …