© 2018, Prof. H.S. Srivastava Foundation for Science and Society. The root bark of Semialarium mexicanum (Miers) Mennega (cancerina) is traditionally used in Mexico to treat cancer. However, there are no studies supporting its use. We evaluated whether S. mexicanum root bark induces cytotoxicity in breast cancer cells to determine if it has potential applications in the treatment of this disease. Extracts of S. mexicanum root bark in petroleum ether, ethanol, and water were obtained by ultrasound-assisted extraction. MTT and WST-1 assays were used to evaluate the cytotoxicity of the extracts toward breast cancer cells (MDA-MB-231 and MCF7), non-tumorigenic breast-derived cells (MCF 10A), and peripheral blood mononuclear cells (PBMCs). For the extract with greatest cytotoxicity, induction of apoptosis and oxidative stress were determined using flow cytometry. The extract was fractionated, and the cytotoxicity of its fractions was evaluated with the four cell types. The fractions were also analyzed by HPLC. Only the petroleum ether extract was cytotoxic for all cell types (MDA-MB-231 > MCF 10A/MCF7 > PBMCs). Cell death occurred by apoptosis, which could be associated with the induction of oxidative stress. Two fractions that were highly cytotoxic for breast cancer cells were obtained from this extract (IC 50 ≤ 4.15 µg/mL for the most active fraction at 72 h). The MCF 10A cells were less affected, while PBMCs were not affected after 72 h of treatment. Pristimerin was identified in both fractions and may be partially responsible for the cytotoxic effect. These results suggest that S. mexicanum root bark has a potential application in breast cancer treatment.