The Electron Spin Resonance (ESR) spectra of radicals obtained from two new potential antitrypanosomal drugs by Trypanosoma cruzi reduction were analyzed. DMPO Spin Trapping was used to investigate the possible formation of free radicals in the trypanosome microsomal system. The Nitro 2 (4-(n-butyl)-1-(5-nitrofurfurylidene)semicarbazide) analogue of Nifurtimox showed better antiparasitic activity than N-oxide 1 (4-(n-butyl)-1-[(7-bromo-N1-oxidebenzo[1,2-c]1,2,5-oxadiazole-5-yl)methylidene]semicarbazide). Only Nitro 2 could produce oxygen redox cycling in T. cruzi epimastigotes. The ESR signal intensities were consistent with the trapping of hydroxyl radical. These results are in agreement with the biological observation that Nitro 2 showed antichagasic activity by an oxidative stress mechanism