Sixteen 5,6-dihydro-2H-pyran-2-ones were evaluated in in vitro assay against trypomastigotes forms of Trypanosoma cruzi, the causative agent of Chagas' disease. A structure-activity relationship study (SAR) allowed us to establish the relevant structural features for the trypanocidal activity of goniothalamin analogues against T. cruzi. In fact, non-natural form of goniothalamin (ent-1) was threefold more potent than the natural one (1). In addition, we have identified analogues 9 and 10 (both displaying S configuration) as the highest potent compounds against T. cruzi with IC50=0.12 and 0.09 mM (IC50 value for crystal violet was 0.08 mM) whereas significantly lower toxicities were observed when these compounds were evaluated under LLC-MK2 lineage cells (1.38 and 4.89 mM, respectively). In addition, epoxides derivatives 12 and ent-12 were shown to be more potent than the corresponding stereoisomers 2 and ent-2 and non-natural argentilactone (ent-3, IC50=0.47 mM) was twofold more potent than natural argentilactone (3, IC50=0.94 mM).