Objective: To evaluate the chemical components of active extract from Cnidoscolus quercifolius root bark and its cytotoxic potential against several tumor strains. Methods: The high-performance liquid chromatography with diode-array detection and 1H and 13C nuclear magnetic resonance spectroscopy of the extract were used to distinguish the existence of possible functional groups in the root bark extract. The in vitro cytotoxic activity of methanol extract on human colon cancer cell lines was evaluated using OVCAR-8, SF-295, HCT116, HL-60 strains and the samples were assessed by 3-(4,5-dimethylthiazol2-yl)-2,5diphenyltetrazolium bromide method. Results: The analysis of nuclear magnetic spectra of the active chloroform fraction revealed the presence of absorptions bands correspondent to a mixture of favelines such as neofavelanone, deoxofaveline or methyl-faveline, which structures were confirmed by ultraviolet spectra upon high-performance liquid chromatography with diode-array detection analysis. The active fraction showed cytotoxic effects in the tested strains, HCT-116, SF-295, OVCAR-8 and HL-60 cells with IC50 of 72 hours ranging from 4.95 to 15.23 μg/mL. Conclusions: The results suggest that the substances present in faveleira (Cnidoscolus quercifolius) root bark extract have a cytotoxic potential against several tumor lines, showing a broader antitumour potential, and in addition no adverse to healthy cells. Therefore, the root bark extract of Cnidoscolus quercifolius has a possibility of use for anticarcinogenic therapies.