In countries where leishmaniasis is endemic, there are not very many treatment alternatives and most options have problems associated with their use. Plants and their natural products constitute good sources of interesting lead compounds that could be potentially active against Leishmania. Chenopodium ambrosioides is a plant that is widely used in popular medicine and its antiparasitic effects have been documented, including the antileishmanial potentialities of Chenopodium oil. The objective of this study was to determine the chemical composition, in-vitro cytotoxicity and antileishmanial activity of essential oils extracted from C. ambrosioides, which received different treatments prior to extraction. The chemical characterization by GC-MS of the three essential oil samples showed similar composition and the major components were alpha-terpinene (17.0-20.7%), p-cymene (20.2-21.1%) and ascaridole (30.5-47.1%). The essential oils exhibited similar antileishmanial activities against intracellular amastigote form, with IC50 values between 4.7 and 12.4 microg/mL. However, a lower cytotoxicity was displayed by the essential oil extracted from fresh green vegetable material, which was statistically different (P < 0.05) from the other samples. This study demonstrated that the prior treatment of plant material did not interfere with the antiparasitic activity of essential oils from C. ambrosioides but did change their cytotoxicity, which should be taken into account in further studies.