Dichloromethane and ethanol extracts of Eupatorium arnottianum were evaluated for invitro leishmanicidal activity. The dichloromethane extract produced 80% and 85% of growth inhibition on both Leishmania mexicana and Leishmania amazonensis promastigotes respectively, at a concentration of 100 μg/mL. At the same concentration, the ethanol extract inhibited the growth of L. mexicana and L. amazonensis promastigotes in 62% and 65% respectively. Bioassayguided fractionation of these extracts led to the isolation and identification of flavonoids nepetin, eupatilin, jaceosidin, eriodictyol and hyperoside as bioactive compounds against both species of Leishmania with IC50 values <10 μg/mL, with nepetin being the most active compound against both Leishmania species (IC50 values <2 μg/mL). On Trypanosoma cruzi epimastigotes, jaceosidin, eupatilin and nepetin were found to be the most active flavonoids, with IC50 values 0.06, 0.89 and 2.94 μg/mL respectively. It was observed by transmission electron microscopy that eupatilin and jaceosidin induced intense cytoplasmic vacuolization on L. mexicana promastigotes, followed by parasite destruction. Nepetin induced a mild vacuolization, although the morphology of parasites was preserved. On T. cruzi epimastigotes, eupatilin and jaceosidin induced a moderate vacuolization and some kinetoplast swelling while nepetin did not induce any significant effects on parasites. At the ultrastructural level, the differential effects of eupatilin and jaceosidin on both trypanosomatids may indicate that these molecules affect selectively certain targets on Leishmania spp. We conclude that plants from the Eupatorium genus could be an important source of compounds (flavonoids) with leishmanicidal and trypanocidal activity, thus constituting an interesting option for the treatment of parasitic diseases.