A new series of cyclohexyl-quinoxaline-7-carboxylate-1,4-di- N -oxide derivatives was synthesized and evaluated in vitro against the trypanosomatids Trypanosoma cruzi and Leishmania mexicana , and the amitochondriates Giardia lamblia, Trichomonas vaginalis, and Entamoeba histolytica . The potential mechanism of action was investigated through molecular docking on T. cruzi and L. mexicana trypanothione reductase (Tc TR and Lm TR, respectively), and on T. cruzi , G. lamblia, and T. vaginalis triosephosphate isomerase (Tc TIM, Gl TIM, and Tv TIM, respectively), and E. histolytica thioredoxin reductase (EhTrxR). Enzyme inhibition assays were performed on recombinant Tc TR, Tc TIM, G l TIM, and Eh TrxR. Compounds 6 , 7 , and 10 exhibited the best trypanocidal activity against T. cruzi trypomastigotes (IC<inf>50</inf> = 1.9, 8.6, and 0.8 µM, respectively), while compound 4 was the most active derivative against T. cruzi intracellular amastigotes (IC<inf>50</inf> = 72.96 µ M). Compounds 6 , 10 , 14 , and 15 exhibited uncompetitive inhibition on Tc TR (Ki = 26, 50, 30, and 146 µ M, respectively). Compounds 7 (IC<inf>50</inf> = 4.99 µ M) and 14 (IC<inf>50</inf> = 65.32 µ M) were the most leishmanicidal agents against QEPS and M379 L. mexicana strains, respectively; additionally, 7 was the most selective (SI = 34.37). On the other hand, compounds 8 and 14 had the best giardicidal, trichomonacidal, and amoebicidal activity (IC<inf>50</inf> ' 0.20 µ M); additionally, compound 13 had a potent trichomonacidal and amoebicidal activity (IC<inf>50</inf> = 0.12 and 0.18 µ M, respectively), while compounds 8 and 14 were the most selective (SI ' 800). Finally, compounds 7 and 14 exhibited broad-spectrum activity, as they showed noteworthy activity against trypanosomatids and amitochondriate organisms. These results encourage the development of more potent and selective antiparasitic agents using the quinoxaline 1,4-di- N -oxide scaffold.