Pre-fractionation and contemporary spectroscopic analysis provides important clues with regard to synergistic and antagonistic effects as well as biological activities in complex multi-mixtures, especially natural products. Phytochemical contents of the Chrysophthalmum montanum root extracts were analyzed by gas chromatography–mass spectrometry and high performance liquid chromatography–time of flight/mass spectrometry. Furthermore, biological activity of root extract was investigated by antiproliferative efficiencies against human uterus carcinoma and rat brain tumor cells in vitro. The biological activity tests were carried out as dose-dependent assay using a multiwell spectrophotometer. 5-Fluorouracil was used as standard anticancer drug. Total 61 lipophilic components were identified quantitatively from the sub-fraction of n-hexane in which saturated fatty acids (33.06%), unsaturated fatty acids (21.03%) and other organic components (43.35%). Twenty three hydrophilic components, 16 phenolic and 7 flavonoid compounds, were quantified in the hydrophilic sub-fractions obtained from the C. montanum roots. Among the hydrophilic components, the vanillic acid was analyzed as 46.463 ± 0.167 mg compound/g dry extract in the sub-fraction dichloromethane. The linoleic acid was analyzed as a substantial component (8.13%) in the sub-fraction n-hexane. The highest antiproliferative activities were obtained from the sub-fraction dichloromethane against HeLa cells (IC 50 = 36.53 μg/mL) and the sub-fraction n-hexane against C6 cells (IC 50 = 11.98 μg/mL). No cytotoxic effect was observed from these sub-fractions.