Background: Ziziphus spina-christi (Z s-c) is traditionally been used as an herbal medicine and is part of Rhamnaceae plants family. Objectives: The probable cytotoxic effect of Z s-c, found in Al Bahah region, on human oral cancer cell lines (SCC-9) was assessed in the present study. Materials and Methods: Bioactive compounds of Z s-c were evaluated using thin layer chromatography, analytical high-performance liquid chromatography and nuclear magnetic resonance spectroscopic analyses. The cytotoxic effect of Z s-c on SCC-9 cells was revealed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The mechanisms involved in the cytotoxicity of Z s-c were investigated in terms of apoptosis, mitochondrial membrane potential, and cell cycle using flow cytometry, while caspase-9 and p53 activity was investigated using the Western Blot analysis. Results: Z s-c has potent variable cytotoxic effect on SCC-9 cells demonstrated through lactate dehydrogenase and comet assay. An effective cell cycle arrest was observed at the G2M phase with apoptotic induction. They significantly induce G2M cell cycle arrest and promote apoptosis in SCC-9 cell line. The expression of caspase-9 and p53 was induced by Z s-c in a dose-dependent manner. Conclusion: Z s-c may be a novel candidate for the development of new natural product-based therapeutic agents against oral cancer.