As a fourth approach of quinoxaline N,N’-dioxides as anti-trypanosomatid agents against T. cruzi and Leishmania, we found extremely active derivatives. The present study allows us to state the correct requirements for obtaining optimal in vitro anti-T. cruzi activity. Derivatives possessing electron-withdrawing substituent in the 2-, 3-, 6-, and 7-positions rendered the most active compounds. With regard to these features, and taking in account their mammal-cytotoxicity, some trifluoromethylquinoxaline N,N’-dioxides have been proposed as candidates for further clinical studies. Consequently, mutagenicity and in vivo analyses were performed with one of the most promising derivatives. In addition, with regard to the mechanism of action studies, it was demonstrated that mitochondrial dehydrogenases are involved in the anti-T. cruzi activity of the most active derivatives.