American trypanosomiasis is a worldwide health problem that requires attention due to ineffective treatment options. We evaluated n-butyl and isobutyl quinoxaline-7-carboxylate 1,4-di--oxide derivatives against trypomastigotes of the strains NINOA and INC-5. An in silico analysis of the interactions of 1,4-di--oxide on the active site of trypanothione reductase (TR) and an enzyme inhibition study was carried out. The n-butyl series compound identified as T-150 had the best trypanocidal activity against trypomastigotes, with a 13% TR inhibition at 44 μM. The derivative T-147 behaved as a mixed inhibitor with Ki and Ki' inhibition constants of 11.4 and 60.8 µM, respectively. This finding is comparable to the TR inhibitor mepacrine (Ki = 19 µM).