OBJECTIVE Fisetin is a yellow-coloring flavonoid that can be found in a wide variety of plants, vegetables, and fruits, such as strawberries, apples, and grapes. It has been shown to have biological activity by targeting different pathways regulating survival and death and to bear antioxidant and anti-inflammatory activity. Fisetin was shown to be cytotoxic on different cancer cell lines and has the ability to kill therapy-induced senescent cancer cells. The aim of the study was to investigate the DNA damaging and cytotoxic potential of fisetin and its ability to enhance the killing effect of temozolomide on glioblastoma cells. METHODS We used LN229 glioblastoma cells and measured survival and apoptosis by flow cytometry, DNA strand breaks by the alkaline comet and γH2AX assay, and the DNA damage response by western blot analysis. RESULTS Fisetin was cytotoxic on glioblastoma cells, inducing apoptosis. In the dose range of 40-80 μM it also induced DNA damage, as measured by the alkaline comet and γH2AX assay, and triggered DNA damage response, as revealed by p53 activation. Furthermore, fisetin enhanced the genotoxic effect of methyl methanesulfonate, presumably due to inhibition of DNA repair processes. When administered together with temozolomide, the first-line therapeutic for glioblastoma, it enhanced cell death, reduced the yield of senescent cells following treatment and exhibited senolytic activity on glioblastoma cells. CONCLUSIONS Data show that high-dose fisetin has a genotoxic potential and suggest that, harnessing the cytotoxic and senolytic activity of the flavonoid, it may enhance the effect of anticancer drugs and eliminate therapy-induced senescent cells. Therefore, it may be useful for adjuvant cancer therapy, including glioblastoma, which is worth to be studied in clinical trials.