Mitonafide (4-nitro-benzoisoquinolinedione) and a number of structural analogs were synthesized and studied in order to determine the structural requirements for inhibition of leishmanial nuclear and kinetoplast topoisomerase II and human topoisomerase II. The structure-activity relationship studies with the mitonafide analogs demonstrated that there was selective targeting of leishmanial nuclear topoisomerase II and human topoisomerase II and differential targeting of kinetoplast over nuclear topoisomerase II in the parasite. Mitonafide analogs appeared to have multiple mechanisms of action leading to death of leishmanias, but several compounds that affected kinetoplast but not nuclear topoisomerase II were not cytotoxic as determined by short-term assays. These studies provide new insight into the differential sensitivities of leishmanial nuclear and kinetoplast topoisomerase II to topoisomerase II-targeting drugs.