Previously, we have identified a series of 5-nitroindazoles with good antiprotozoal activities, against Trypanosoma cruzi epimastigotes and Trichomonas vaginalis. Most of them have shown very low unspecific toxicity on macrophage cell lines. In the present work, we assayed these compounds on T. cruzi bloodstream trypomastigotes and Leishmania promastigotes (Leishmania amazonensis, Leishmania braziliensis and Leishmania infantum). Derivatives 1, 2, 7 and 8 displayed remarkable trypanocidal activity (>80% lysis) equivalent to gentian violet. Derivatives 2 and 10, as Pentamidine, caused the complete lysis of promastigotes of Leishmania. An oxidative stress-mediated mechanism of action was confirmed for derivatives 1, 10 and 12 on T. cruzi epimastigotes. Supported by the in vitro activities, derivatives 1 and 2 were submitted to in vivo assays using an acute model of Chagas' disease and a short-term treatment. None of the animals treated with derivatives 1 and 2 died, unlike the untreated control and Benznidazole groups.