Human DNA topoisomerase II (Top2) is a promising target for cancer treatment. To overcome the limitations of known Top2 inhibitors, novel Top2 catalytic inhibitors with new scaffolds were identified by structure-based virtual screening. In particular, compound 8 showed good in vitro antiproliferative activity with a broad spectrum. Top2-mediated cleavage assay and molecular modeling rationalized the mode of action. The new Top2 inhibitors are considered as good starting points for further hit-to-lead optimization in anticancer drug discovery.