Abstract Hymenocrater longiflorus was collected from northern Iraq, and the chemical composition and antioxidant and cytotoxic activities of this plant were investigated. Ten compounds detected by HPLC-ESI/MS were identified as flavonoids and phenolic acids. The free radical scavenging activity of the 70% methanol extract was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The antioxidant activities of the extract may be attributed to its polyphenolic composition. The cytotoxicity of the plant extract against the osteosarcoma (U2OS) cell line was assessed with the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The extract significantly reduced the viability of cells in a concentration- and time-dependent manner. Cells were arrested during the S-phase of the cell cycle, and DNA damage was revealed by antibodies against histone H2AX. The apoptotic features of cell shrinkage and decrease in cell size were also observed. Western blot analysis revealed cleavage of poly (ADP-ribose)-polymerase 1 (PARP-1), in addition to increases in the proteins p53, p21, and γ-H2AX. Collectively, our findings demonstrate that the H. longiflorus extract is highly cytotoxic to U2OS cells, most likely due to its polyphenolic composition.