The present study investigates the effect of nickel oxide nanoparticles (NiO-NPs) on C6 glioma cells and develops a method for preparing NiO. Plant-based materials (leaf extract) can produce NPs efficiently and economically. Therefore, we developed NiO-NPs from Terminalia chebula leaf extract to reduce C6 glioblastoma cell proliferation. The structural, optical, and antimicrobial properties of NiO-NPs were investigated. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, Acridine orange/ethidium bromide dual staining, Hoechst 33342, and Rh123 staining were used to evaluate nuclear changes and mitochondrial membrane potential (MMP) in C6 glioblastoma cells. X-ray diffraction analyses revealed the cubic structures of the synthesized NiO-NPs, field emission scanning electron microscope analysis revealed polygonal NiO-NPs and an energy-dispersive X-ray spectrometer confirmed the high purity of the synthesized NiO-NPs. V. cholera, S. pneumonia, S. aureus, B. subtilis, P. aeruginosa, K. pneumonia, and C. albicans were sensitive to NiO-NPs. When NiO-NPs were applied at lower concentrations to rat glioblastoma C6 cells, they dose-dependently inhibited viability and induced apoptosis. Our findings show that NiO-NPs exhibit altered MMP and nuclear integrity. In this study, NiO-NPs were synthesized using T. chebula leaf extract, which has antiproliferative properties, and NiO-NPs increased cell cytotoxicity in C6 cells. Further exploration of NiO-NPs in glioblastoma animal models should be investigated.