<b><i>Introduction:</i></b> Few published in vitro studies have shown antitumor drug action or possible synergistic effects of fluoroquinolones. To assess the potential role of combination therapy, cytotoxic effects of ciprofloxacin and epirubicin alone and in combination were determined. <b><i>Material and Methods:</i></b> Human urothelial cancer cell lines HT1197 and HT1376 were exposed in vitro for 1 h to different concentrations of epirubicin (0.02–2 mg/ml) and for 72 h to ciprofloxacin (0.004–0.8 mg/ml). Cytotoxicity was determined using the microculture tetrazolium assay and flow cytometry. Synergistic cytotoxic effects were determined by calculating combination indices. <b><i>Results:</i></b> Median effect concentrations of epirubicin for HT1376 and HT1197 cells were as low as 124 and 117 µg/ml, respectively. Ciprofloxacin-treated cells exhibited profound cytotoxic effects at concentrations of 50–100 µg/ml, which is far below the intravesical concentration reached by standard oral application. In addition, a pronounced synergistic effect was found when the two treatments were combined. <b><i>Conclusions:</i></b> This study provides evidence that ciprofloxacin and epirubicin exhibit synergistic cytotoxic effects in vitro. After confirmatory animal experiments, future clinical studies of adjuvant chemotherapy after transurethral bladder resection may include treatment arms with combinations of fluoroquinolones based on the observed synergistic effects to reduce both side effects and costs.