Chagas' disease is a neglected tropical disease caused by Trypanosoma cruzi and constitutes a serious public health problem for Latin America. Its unsatisfactory chemotherapy stimulates the search for novel antiparasitic compounds. Amidines and related compounds exhibit well-known activity towards different microbes including T. cruzi. In this vein, our present aim was to evaluate the biological effect of 10 novel structurally related amidines in vitro against bloodstream and intracellular forms of the parasite as well as their potential toxicity on cardiac cell cultures. Our results show that although active against the extracellular forms, with some of them like DB2247 being 6-fold more effective than benznidazole and displaying very low toxicity (>96 μm), none presented superior trypanocidal effect against intracellular forms as compared with the reference drug. These results may be due to differences in susceptibility profiles related to distinct uptake/extrusion mechanisms and cellular targets between bloodstream and amastigote forms. The present study adds to the knowledge base for the future design of novel amidines that may provide promising activity against T. cruzi.