The anthocyanin-rich extract (ARE) of the fruit from Pourouma cecropiifolia, a tropical plant native to the Amazon region, showed moderate cytotoxicity toward different cancer cell lines when evaluated by MTT assays. This extract was fractionated using Sephadex LH-20 chromatography to obtain three fractions (F1−F3), the composition of which was analyzed by HPLC-PDA and LC-ESI/MS. F1 was composed primarily of the monomeric anthocyanins delphinidin-3-O-β-glucopyranoside, cyanidin-3-O-β-glucopyranoside, and cyanidin-3-O-(6′′-malonyl)glucopyranoside. F2 contained the isomeric flavonols quercetin 3-O-α-rhamnopyranosyl-(1→6)-β-galactopyranoside and quercetin 3-O-α-rhamnopyranosyl-(1→6)-β-glucopyranoside, the structures of which were confirmed by 1H and 13C NMR. F3 contained polymeric pigments, which were analyzed using tandem ESI/MS with an ion trap-TOF. The structures of two proanthocyanidin and two flavanol-anthocyanin condensed pigments were suggested on the basis of their MSn fragmentation patterns. After cell viability assays were performed, only fraction F3 showed a cell growth-inhibitory effect similar to the one found for ARE. F3 significantly reduced the viability of HEp-2 larynx, MKN-45 gastric carcinoma, and MCF-7 breast cancer cells; in contrast, the pure compounds did not show promising cytotoxicity toward the cancer cells evaluated.