Fungal topoisomerase II (TopoII) has been identified as essential for viability. Thus, our research aimed to investigate the potential of fungal TopoII as a novel target for antifungal chemotherapy. We conducted studies on eleventh antitumor compounds targeting human topoisomerase II, either approved by the U.S. Food and Drug Administration (FDA) or currently under clinical trials to evaluate their potential for use in other therapeutic applications. While most of the compounds we analyzed are potent inhibitors of yeast TopoII, only a few exhibited antifungal activity. Idarubicin emerged as the most potent compound effectively inhibiting the growth of five reference fungal strains as well as clinical Candida glabrata fluconazole-resistant cells. Antifungal activity of this compound corresponded with its very high yeast TopoII inhibitory effectiveness. Additionally, idarubicin ability to be effectively accumulated into fungal cells is crucial for yeast TopoII targeting. Idarubicin, epirubicin, and bisantrene appeared to be even more effective inhibitors of yeast enzyme than its human counterpart. In fungal cells idarubicin exhibited a multifaceted mechanisms of action, including nuclear DNA fragmentation, disruption of mitochondrial network architecture and mitochondrial DNA aggregation as well as oxidative stress induction. Our results indicate that fungal topoisomerase II targeting is worth considering in antifungal treatment and the reported drugs may serve as a starting point for the reinnovation of a new molecule.