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Zinc oxide nanoparticles (ZnO NPs) have become a highly regarded substance in various industries especially biologically synthesized ZnO NPs due to their adherence to the principles of green chemistry. However, concerns have been raised regarding the potential cytotoxic effects of ZnO NPs on biological systems. This study aimed to investigate and compare the cytotoxicity of ZnO NPs that were synthesized through chemical (C–ZnO NPs) and green approach using Musa acuminata leaf aqueous extract (Ma-ZnO NPs) on Vero cells.Characterization of ZnO NPs through Uv–Vis, FESEM, EDX, XRD, FTIR and XPS confirmed the successful synthesis of C- and Ma-ZnO NPs. MTT and ROS assays revealed that C- and Ma-ZnO NPs induced a concentration- and time-dependent cytotoxic effect on Vero cells. Remarkably, Ma-ZnO NPs showed significantly higher cell viability compared to C–ZnO NPs. The corelation of ROS and vell viability suggest that elevated ROS levels can lead to cell damage and even cell death. Flow cytometry analysis indicated that Ma-ZnO NPs exposed cells had more viable cells and a smaller cell population in the late and early apoptotic stage. Furthermore, more cells were arrested in the G1 phase upon exposure to C–ZnO NPs, which is associated with oxidative stress and DNA damage caused by ROS generation, proving its higher cytotoxicity than Ma-ZnO NPs. Similarly, time-dependent cytotoxicity and morphological alterations were observed in C- and Ma-ZnO NPs treated cells, indicating cellular damage. Furthermore, fluorescence microscopy also demonstrated a time-dependent increase in ROS formation in cells exposed to C- and Ma-ZnO NPs.In conclusion, the findings suggest that green ZnO NPs possess a favourable biocompatibility profile, exhibiting reduced cytotoxicity compared to chemically synthesized ZnO NPs on Vero cells. These results emphasize the potential of green synthesis methods for the development of safer and environmentally friendly ZnO NPs.