Background: Cnidoscolus quercifolius is a Brazilian medicinal plant often found in the Caatinga biome. Previous studies have described several pharmacological properties for this plant, including antiproliferative effect. However, there are still few pharmacological and phytochemical reports involving this plant. Objective: In this report, it was described the cytotoxic effect of extract and fractions obtained from the leaves of C. quercifolius. It was also reported for the first time the identification of two flavonoids in this species. Materials and Methods: Ethanol extract (EE) and fractions hexane, chloroform-Fr, ethyl acetate (AcOEt-Fr) and methanol (MeOH-Fr) were evaluated against prostate (PC3 and PC3-M) and breast (MCF-7) cancer cell lines. A preliminary phytochemical analysis was performed by thin layer chromatographic, while the content of total phenolic compounds and flavonoids was determined by colorimetric assays. EE and bioactive fraction (AcOEt-Fr) were selected for analysis by high-performance liquid chromatography-diode-array detector (HPLC-DAD). Results: Phytochemical analysis revealed that the samples were positive for the presence of several classes of secondary metabolites, mainly phenolic derivatives and flavonoids. EE and AcOEt-Fr presented the highest phenolic and flavonoid content. HPLC-DAD analysis of EE and AcOEt-Fr allowed the identification of two flavonoids (rutin and apigenin) not yet described for this species. Concerning the cytotoxicity evaluation, only AcOEt-Fr demonstrated a strong cytotoxic effect against all cell lines, presenting the half maximal inhibitory concentration values between 15.75 and 46.97 μg/ml. Conclusion: The results suggest that flavonoids may play an important role in the cytotoxic effect observed for this species. In addition, this report contributed to the phytochemical knowledge of the species through the identification and quantification of flavonoids.