We evaluated the chemical composition, antioxidant activity, and antitumor potential of a fraction that was isolated from Stryphnodendron adstringens (barbatimo) leaf aqueous extract. Fraction is composed by gallic acid, procyanidin dimer B1, and (-)-epicatechin-3-O-gallate and it exhibits antioxidant and cytotoxic activities. Fraction was cytotoxic against two human breast cancer cell lines, ER (+) and MCF-7 and the triple-negative, MDA-MB-435. The sulforhodamine B assay showed that, as compared to normal control cells, the fraction significantly (P < 0.05) decreased cancer cell viability. The morphological alterations noted in the treated cancer cells were cell rounding-up, shrinkage, and nuclear condensation reduction of cell diameter and length. Treatment with fraction increased cancer cell expression of Bax, caspase-9, active caspase-3, caspase-8, LC-3, and beclin-1 and decreased Bcl-2, caspase-3, and pro-caspase-8 expression. Altogether, fraction is cytotoxic to both breast cancer cell lines, induces cell death, and its mechanism of action seems to include the induction of apoptosis. Our data support a positive role of the fraction as a chemopreventive agent for antineoplastic drug development.