AIM : To determine the computer-predicted anticancer activity of mupirocin and to compare its activities with those determined for another polyene antibiotic, batumin. MATERIALS AND METHODS : Molecular docking, cytotoxicity assays, cell microscopy and cell cycle progression were studied in cancer and nontumorigenic cell lines. RESULTS AND CONCLUSION : Cytotoxicity of mupirocin against several cancerous cell lines was detected with the highest one (IC50 = 5.4 μg/ml) against melanoma cell line. The profile of cytotoxicity of mupirocin was similar to that reported for batumin. Nevertheless, the morphology of cells treated with these antibiotics and alterations in cell cycle progression suggested possible dissimilarity in their mechanisms of action. Selective cytotoxicity of mupirocin against melanoma cells potentiates further studies to discover nontoxic drugs for melanoma prevention.