The purpose of this study is to evaluate the cytotoxic activity of Cichorium intybus herb as well as to identify the molecular mechanism of the cytotoxicity. In addition, it aims to investigate its phytoconstituents that are responsible for the bioavailability. The cytotoxic activity of Cichorium intybus herb was assayed by SRB (Sulforhodamine B) assay against ovarian cancer cell line (SKOV-3), liver cancer cell line (HepG2) and prostate cancer cell line (PC-3). The effect on tubulin polymerization was studied to identify the mechanism of cytotoxicity. The binding affinity to the target molecule was examined by docking study. In addition, two flavonoidal compounds were isolated and identified by different spectroscopic methods. The results showed that the methanol extract of Cichorium intybus herb as well as the isolated compounds (myricetin and pinobanksin) possessed a potent cytotoxicity against HepG2 (IC50 =0.95, 4.26 and 7.23 μg/mL), respectively, moderate cytotoxicity against PC-3 (IC50 =25.34, 36.24 and 42.53 μg/mL), respectively, and weak cytotoxicity against SKOV-3 (IC50 >100 μg/mL) for all tested samples. Molecular docking analysis confirmed that both of the isolated compounds showed high binding affinity to colchicine binding site of tubulin microtubules, supported the high cytotoxicity of these compounds.