Chagas disease (CD) is caused by the intracellular protozoan parasite Trypanosoma cruzi and affects more than 10 million people in poor areas of Latin America. There is an urgent need for alternative drugs with better safety, broader efficacy, lower costs and shorter time of administration. Thus the biological activity of viniconazole, a chloroaryl-substituted imidazole was investigated using in vitro and in vivo screening models of T. cruzi infection. Ultrastructural findings demonstrated that the most frequent cellular damage was associated with plasma membrane (blebs and shedding events), Golgi (swelling aspects) and the appearance of large numbers of vacuoles suggesting an autophagic process. Our data demonstrated that although this compound is effective against bloodstream and intracellular forms (16 and 24 μM, respectively) in vitro, it does not present in vivo efficacy. Due to the urgent need for novel agents against T. cruzi, the screening of natural and synthetic products must be further supported with the aim of finding more selective and affordable drugs for CD.