Chagas disease is a health problem that affects millions of persons, currently Nifurtimox (Nfx) and Benznidazole (Bz) are the unique drugs to treat it. However, these drugs produce adverse effects and high toxicity, which has motivated the search for new candidate drugs. Based on reports about the extensive biological activity of steroidal nitrate esters, in this study three nitrate esters steroids (1b, 2b and 4b) were synthetized and characterized from Dehydroepiandrosterone (DHEA, 1a), 19-hydroxy-DHEA (2a), and Androst-5-en-3β,17β-diol (4a), respectively. In addition, compounds 3a and 3b were obtained by introducing an α-ethynyl and a β-hydroxyl groups at position 17 of 2b and further nitration of the hydroxyl group. The trypanocidal activity of these steroids was evaluated in vitro against the epimastigote stage of two T. cruzi strains, Ninoa and TH, and their cytotoxicity over J774.2 macrophage cell line was assayed. Compounds 3a, 3b, and 4a shown higher trypanocidal activity than Bz and Nfx against epimastigotes of Ninoa strain, whereas DHEA (1a) and its nitrate derivative 1b showed higher activity than the reference drugs against the TH strain epimastigote. None of the compounds showed activity in the ex vivo assays against the blood trypomastigote of both strains. Interestingly, the selectivity index of Androst-5-en-3β,17β-diol 4a was almost twice the value of Nfx and 50 times more than Bz, against Ninoa and TH strains, respectively. Therefore, compound 4a could represent a valuable starting point toward the optimization of steroid derivatives as trypanocidal agents.