Background and Objective: Glioma is known to be highly resistant to radiotherapy and chemotherapy. Therefore, new therapeutic agents for glioma are being extensively researched. In this study, we aimed to investigate anti-tumoural effects of Xanthium strumarium L. (Asteraceae family) extracts and its pure constituents on C6 glioma cells. Materials and Methods: In this study, cytotoxicity, lipid peroxidation, apoptotic effect, phosphatidylserine externalization, cell cycle analysis, invasion, kinase activity, COX-2 expression and micronucleus tests were used. Results: As a result of IR, 1H-NMR, 13C-NMR, 1D and 2D NMR analysis, 5 known compounds were characterized as xanthinosin (1), stigmasterol (2), xanthatin (3), xanthinin (4) and xanthanol (5). Among them, the most cytotoxic xanthanolide was xanthinin (4) with an IC50 value of 7.5 μM. It was thought that this effect may be due to the oxidative damage and protein kinase activity of this compound. While xanthinosin (1) and xanthanol (5) showed the selective cytotoxic effect on C6 glioma cells with low IC50 values (22.46 and 40.12 μM, respectively), it was determined that their toxicity on Human Umbilical Vein Endothelial (HUVEC) cells was lower (IC50 >75 μM). Conclusion: It was concluded that xanthanolides isolated from X. strumarium could be used as target molecules in future studies as chemotherapeutic agents on glioma.