Background: Combination therapy has generated remarkable motivation in the clinical setting since it boosts the therapeutic potential of anticancer drugs. Glioblastoma multiforme (GBM) is the most common and aggressive malignant brain tumor which affects patients in all ages, and being principally resistant to treatment. Methods: In this study, temozolomide as a standard chemotherapeutic drug for Human GBM, was combined with ciprofloxacin to know whether ciprofloxacin can potentiate the cytotoxic effects of temozolomide. Glioblastoma A-172 cell line was exposed to ciprofloxacin and temozolomide either alone or in combination for 24, 48 and 72 h. Cytotoxicity was measured by MTT assay. Results: Ciprofloxacin and temozolomide induced tumor cell death in a dose-dependent manner with an IC50 value of 259.3 μM for ciprofloxacin and 62.5 μM for temozolomide in 72 h. These values shifted to 22.8 μM for ciprofloxacin and 8.6 μM for temozolomide in the presence of IC50 of the other drug. The combination-index (CI) values were found less than 1. Conclusion: These results indicate synergism across a broad range of concentrations of ciprofloxacin and temozolomide in glioblastoma tumor cell line and that CPF increased the anti-tumor cytotoxic effect of TMZ in glioblastoma A-172 cell line.