Croton sphaerogynusis a shrub from the Atlantic Rain Forest in southeastern Brazil. A lyophilized crude EtOH extract from leaves ofC. sphaerogynus, obtained by maceration at room temperature (seven days), was suspended in methanol and partitioned with hexane. The purified MeOH phase was fractionated over Sephadex LH-20 yielding five fractions (F1–F5) containing flavonoids, as characterized by HPLC-DAD and HPLC-MS analyses. The antiproliferative activity of the crude EtOH extract, MeOH and hexane phases, and fractionsF1–F5was evaluated onin vitrocell lines NCI-H460 (nonsmall cell lung), MCF-7 (breast cancer), and U251 (glioma). The MeOH phase showed activity (mean log GI₅₀0.54) higher than the hexane phase and EtOH extract (mean log GI₅₀1.13 and 1.19, resp.).F1exhibited activity against NCI-H460 (nonsmall cell lung) (GI₅₀1.2 μg/mL), which could be accounted for the presence of flavonoids and/or diterpenes.F4showed moderate activity (mean log GI₅₀1.05), whileF5showed weak activity (mean log GI₅₀1.36). It is suggested that the antiproliferative activity of the crude EtOH extract and MeOH phase is accounted for a synergistic combination of flavonoids and diterpenes.