OBJECTIVES: As part of a search for new therapeutic opportunities to treat chagasic patients, in vitro efficacy studies were performed to characterize the activity of five novel arylimidamides (AIAs) against Trypanosoma cruzi. METHODS: The trypanocidal effect against T. cruzi was evaluated by light microscopy through the determination of IC₅₀ values. Cytotoxicity was determined by MTT assays against mouse cardiomyocytes. RESULTS: Our data demonstrated the trypanocidal efficacy of these new compounds against bloodstream trypomastigotes and intracellular amastigotes, exhibiting IC₅₀ values ranging from 0.015 to 2.5 and 0.02 to0.2 μM, respectively. One of the compounds, DB745B, was also highly active against a broad panel of isolates, including those naturally resistant to benznidazole. DB745B showed higher in vitro efficacy than the reference drugs used to treat patients (benznidazole IC₅₀= 12.94 μM) and to prevent blood bank infection (gentian violet IC₅₀= 30.6 μM). CONCLUSIONS: AIAs represent promising new chemical entities against T. cruzi and are also potential trypanocidal agents to prevent transfusion-associated Chagas' disease.