Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, a neglected tropical disease that is endemic in Latin America and spreading worldwide due to globalization. The current treatments are based on benzni dazole and nifurtimox; however, these drugs have important limitations and limited efficacy during the chronic phase, reinforcing the necessity of an alternative chemotherapy. For the last 30 years, our group has been evaluating the biological activity of naphthoquinones and derivatives on T. cruzi, and of the compounds tested, N1, N2 and N3 were found to be the most active in vitro. Here, we show the synthesis of a novel β-lapachone derived naphthoimidazolium named N4 and assess its activity on T. cruzi stages and the mechanism of action. The new compound was very active on all parasite stages (IC50/24 h in the range of 0.8–7.9 μM) and had a selectivity index of 5.4. Mechanistic analyses reveal that mitochondrial ROS production begins after short treatment starts and primarily affects the activity of complexes II-III. After 24 h treatment, a partial restoration of mitochondrial physiology (normal complexes II-III and IV activities and controlled H2O2 release) was observed; however, an extensive injury in its morphology was still detected. During treatment with N4, we also observed that trypanothione reductase activity increased in a time-dependent manner and concomitant with increased oxidative stress. Molecular docking calculations indicated the ubiquinone binding site of succinate dehydroge nase as an important interaction point with N4, as with the FMN binding site of dihydroorotate dehydrogenase. The results presented here may be a good starting point for the development of alternative treatments for Chagas disease and for understanding the mechanism of naphthoimidazoles in T. cruzi.