With continuously rising morbidity and mortality, the scientists focus their efforts to outcome novel therapeutics due to improve cancer patients’ survival. The major undesirable features of conventional therapies were low selectivity, high toxicity, drug resistance, and economic expenses. Opposite, natural compounds of medicinal plants play a crucial role in treatment of several diseases. Currently, many studies investigated the potential of secondary metabolites to fight cancer cells through enhanced apoptotic cell death or energy production pathway as a targeted and smart therapy strategies. The study aimed to evaluate and investigate of antitumor activity of Citrullus colocynthis fruit oil extraction against cancer and normal cell lines. AMGM-5 human glioblastoma and HBL-100 human breast epithelial cells were used to determination cytotoxic effect of C.colocynthis oil by MTT assay and detection of IC50 dose. Furthermore, this study evaluated apoptosis using fluorescent staining. In addition, we investigated of cell migration, invasion, and rapid rate proliferation of cancer cells by wound healing assay. The results showed significant impacts of essential oil accompanied by toxicity, apoptosis, and inhibition of cell proliferation, migration, and invasion against cancer cells, but, normal cells not affected. Our findings suggest natural compounds have possible cancer drugs without side effect on normal cells.