Chagas disease is a parasitic infection affecting millions of people across Latin America, imposing a dramatic socioeconomic burden. Despite the availability of drugs, nifurtimox and benznidazole, lack of efficacy and incidence of side-effects prompt the identification of novel, efficient, and affordable drug candidates. To address this issue, one strategy could be probing the susceptibility of <i>Trypanosoma</i> parasites toward NADP-dependent enzyme inhibitors. Recently, steroids of the androstane group have been described as highly potent but nonselective inhibitors of parasitic glucose-6-phosphate dehydrogenase (G6PDH). In order to promote selectivity, we have synthesized and evaluated 26 steroid derivatives of epiandrosterone in enzymatic assays, whereby 17 compounds were shown to display moderate to high selectivity for <i>T. cruzi</i> over the human G6PDH. In addition, three compounds were effective in killing intracellular <i>T. cruzi</i> forms infecting rat cardiomyocytes. Altogether, this study provides new SAR data around G6PDH and further supports this target for treating Chagas disease.