The present work reports the synthesis and biological activity of a series of 14 benzimidazole derivatives designed to act on the enzyme triosephosphate isomerase of Trypanosoma cruzi (TcTIM). This enzyme is involved in the metabolism of glucose, the only source of energy for the parasite. In this study, we found four compounds that inhibit TcTIM moderately and lack inhibitory activity against human TIM (HsTIM). In vitro studies against T. cruzi epimastigotes showed two compounds that were more active than the reference drug nifurtimox, and these presented a low cytotoxic effect in mouse macrophages (J744 cell line).