Calothrixin A (<b>CAA</b>) is a dual Topo I and II inhibitor but exhibits poor antiproliferative activities and water solubility. Herein, a library of novel <b>CAA</b> analogues was synthesized. Among them, compound <b>F16</b> exhibited superior water solubility (>5 mg/mL) as compared to <b>CAA</b> (<5 μg/mL). The mechanism of action studies confirmed that <b>F16</b> acted as a dual Topo I and II poison. Furthermore, <b>F16</b> displayed potent antiproliferative activities against high Topo I and II expression cell lines A375 and HCT116, with IC<sub>50</sub> values of 20 and 50 nM, respectively. In xenograft models, <b>F16</b> reduced the tumor growth at a dose of 10 or 20 mg/kg without apparent effect on the mouse weight, while the clinically used Topo II inhibitor <b>VP-16</b> dramatically reduced the mouse weight. Collectively, our data demonstrated that <b>F16</b> could be a promising lead for the development of novel dual Topo I and II antitumor agents.