Abstract This study investigated the effects of the flavonoids 5-hydroxy-7,4′-dimethoxyflavone, casticin, and penduletin, isolated from Croton betulaster Müll Arg., Euphorbiaceae, a plant utilized in popular medicine in Brazil, on the growth and viability of the human glioblastoma cell line GL-15. We observed that 5-hydroxy-7,4′-dimethoxyflavone and casticin were not toxic to GL-15 cells after 24 h of exposure. However, casticin and penduletin inhibited the metabolic activity of glioblastoma cells significantly at a concentration of 10 µM (p ≤ 0.05). Flavonoids casticin and penduletin also induced a significant and dose-dependent growth inhibition beginning at 24 h of exposure, and the most potent flavonoid was penduletin. It was also observed that penduletin and casticin induced an enlargement of the cell body and a reduction of cellular processes, accompanied by changes in the pattern of expression of the cytoskeletal protein vimentin. Signs of apoptosis, such as the externalization of membrane phosphatidyl serine residues, nuclear condensation, and fragmentation, were also detected in cells treated with 50–100 µM flavonoids. Our results indicate that flavonoids extracted from C. betulaster present antitumoral activity to glioblastoma cells, with penduletin proving to be the most potent of the tested flavonoids. Our results also suggest that these molecules may be promising supplementary drugs for glioblastoma treatment.