Chagas disease, caused by Trypanosoma cruzi, is an important cause of mortality and morbidity in Latin America. There are no vaccines available, the chemotherapy used to treat this illness has serious side effects and its efficacy on the chronic phase of disease is still a matter of debate. In a search for alternative treatment for Chagas disease, essential oils extracted from traditional medicinal plants Lippia sidoides, Lippia origanoides, Chenopodium ambrosioides, Ocimum gratissimum, Justicia pectorales and Vitex agnus-castus were investigated in vitro for trypanocidal and cytotoxic activities. Essential Oils were extracted by hydrodistillation and submitted to chemical analysis by gas chromatography/mass spectrometry. The concentration of essential oils necessary to inhibit 50% of the epimastigotes or amastigotes growth (IC(50)) and to kill 50% of trypomastigote forms (LC(50)) was estimated. The most prevalent chemical constituents of these essential oils were monoterpenes and sesquiterpenes. All essential oils tested demonstrated an inhibitory effect on the parasite growth and survival. L. sidoides and L. origanoides essential oils were the most effective against trypomastigote and amastigote forms respectively. No significant cytotoxic effects were observed in mouse peritoneal macrophages incubated with essential oils which were more selective against the parasites than mammalian cells. Taken together, our results point towards the use of these essential oils as potential chemotherapeutic agent against T. cruzi.