Some species of the Baccharis genus have been shown to possess important biomedical properties, including cytotoxic activity. In this study, we examined the cytotoxic effect of methanol extract from Baccharis obtusifolia (Asteraceae) in cancer cell lines of prostate (PC-3), colon (RKO), astrocytoma (D-384), and breast (MCF-7). The methanolic extract displayed the largest substantial cytotoxic effect in lines of colon cancer (RKO) and cerebral astrocytoma (D-384). Chromatographic purification of the B. obtusifolia methanolic extract led to the isolation and identification of 5,4′-dihydroxy-7-methoxyflavone (1) and 5-hydroxy-7,4′-dimethoxyflavone (2) compounds of the flavonoid type.