Trypanosomiasis and leishmaniasis pose major public health threats for many countries, particularly those in sub-Saharan Africa and South America. In the present study, we evaluated the in vitro antiprotozoal activity of three irregular, linear sesquiterpene lactones recently isolated from Greek Anthemis auriculata, namely anthecotulide (1), 4-hydroxyanthecotulide (2) and 4-acetoxyanthecotulide (3). Trypomastigote forms of Trypanosoma brucei rhodesiense and T. cruzi as well as axenic amastigotes of Leishmania donovani were used for testing. The cytotoxic potential of the compounds was also assessed against mammalian (rat) skeletal myoblasts (L6 cells). All compounds showed potent trypanocidal and leishmanicidal activity. 4-Hydroxyanthecotulide (2) appeared to be the most active compound against all parasites, particularly towards T. b. rhodesiense (IC(50) 0.56 microg/ml), whereas 4-acetoxyanthecotulide (3) was the least active. All three metabolites possessed toxicity on mammalian cells, which might limit their use as antiprotozoal agents.