Nanoparticles, smaller than 100 nm are synthesized by chemical and physical methods. Biological synthesis of nanoparticles is very popular in science recently. The aim of the study is green synthesis of zinc oxide nanoparticles (ZnO NPs) using the lichen extract (Ramalina fraxinea) and investigating the cytotoxic effects of ZnO NPs on human neuroblastoma cells (SHSY-5Y). Despite the widespread use of ZnO NPs, a limited number of studies have investigated the neurobiological effects of ZnO NP. Therefore, we tested the neurotoxic effect of green synthesized ZnO NPs administration and its neuroprotective effect against hydrogen peroxide-induced cell damage on SHSY5Y human neuroblastoma cell line. The absorbance peak of the ZnO NPs was detected by UV- visible spectroscopy (UV-Vis) at 330 nm. The average diameter of ZnO NPs was measured as about 21 nm by Scanning Electron Microscope (SEM) and Field Emission Scanning Electron Microscope (FE-SEM) images. According to X-ray Diffraction (XRD) diagram, ZnO NPs were hexagonal in structure. The peaks observed in the Fourier Transform Infrared (FT-1R) test showed functional groups in the structure of the nanoparticles. According to our results, ZnO NPs may have beneficial effects at the low concentrations while neurotoxic effects at the higher doses in SH-SY5Y. In addition, we indicate that hydrogen peroxide-induced cell death could not reverse by ZnO NPs and its higher doses potentiated the neurodegenerative effect of hydrogen peroxide. In conclusion, here we report that ZnO NPs, widely used in various products, may have beneficial or harmful effects in a dose-dependent manner and play a role in neuropsychiatric disease, especially neurodegenerative diseases. This is the first study dealing with neurotoxicity on SHSY-5Y of Ramalina fraxinea extract based ZnO NPs.