New more selective and potent drugs are urgently need to treat Chagas disease (CD). Among the many synthetic compounds evaluated against Trypanosoma cruzi, aromatic amidines (AAs) and especially arylimidamides (AIAs) have potent activity against this parasite. Presently, the effect of four mono-amidines (DB2228, DB2229, DB2292 and DB2294), four diamidines (DB2232, DB2235, DB2251 and DB2253) and one AIA (DB2255) was screened in vitro against different forms (bloodstream trypomastigotes – BT and intracellular forms) and strains from discrete typing unit (DTU) I and VI of T. cruzi and their cytotoxic profile on mammalian host cells. Except for DB2253, all molecules were as active as benznidazole (Bz), resulting in 50% of reduction in the number of alive BT, with EC50 ranging from 2·7 to 10·1 µ
m after 24 h of incubation. DB2255 was also the most potent against amastigotes (Tulahuen strain) showing similar activity to that of Bz (3 µ
m). In silico absorption, distribution, metabolism, excretion and toxicity analysis demonstrated probability of human intestinal adsorption, while mutagenicity and inhibition of hERG1 were not predicted, besides giving acceptable predicted volumes of distribution. Our findings contribute for better knowledge regarding the biological effect of this class of aromatic molecules against T. cruzi aiming to identify novel promising agent for CD therapy.