We aimed to determine the cytotoxic and immunomodulatory effects of hydroalcoholic extracts of the roots and aerial parts of Ebenus boissieri (EB) on breast cancer MDA-MB231 cells and the non-cancerous human embryonic kidney cell line, 293T. Cell viability was determined by MTT assay, trypan blue exclusion, and Live/Dead Viability/Cytotoxicity assay. Apoptosis was evaluated by measuring the activity of caspase-2, 3, 6, 8, and 9. Tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma release was assayed by ELISA, and protein expression of caspase-3, TNF-alpha, and IFN-gamma was determined by western blot. The results of this study revealed that MDA-MB231 cell viability was reduced in a dose-dependent manner by the aerial and root extract of EB at 72 h with a half-maximal inhibitory concentration (IC50) of 41.1 +/- 2.76 and 65 +/- 1.09 mu g/mL, respectively. In contrast, neither the aerial nor the root extracts of this plant inhibited the proliferation of 293T cells at doses up to 1000 mu g/mL. There was a time-dependent increase in caspase activity, especially caspase-3 and caspase-9. The levels of TNF-alpha and IFN-gamma significantly increased in MDA-MB231 cells treated with aerial extract. In conclusion, the extracts of EB induced apoptosis in breast cancer cells by altering the levels of caspases, TNF-alpha, and IFN-gamma. The components and precise modes of action of EB have not yet been determined. However, potential antitumor and immunomodulatory activity was observed along with selectivity against cancer cells in vitro, suggesting that hydroalcoholic extracts of this plant are worthy of additional study.