The trypanocidal effect of six lignan lactones, (−)-cubebin (1), (−)-O-methyl cubebin (2), (−)-O-benzyl cubebin (3), (−)-6,6′-dinitrohinokinin (4), (−)-hinokinin (5) and dimethoxymorelensin (6), previously synthesized by our research group, was evaluated in vitro and in vivo. The compounds with higher anti-epimastigote activity were screened against intracellular amastigote of Trypanosoma cruzi. Among these, compound 5 was selected to be assayed in vivo. It was observed that compounds 5, 6 and 2 showed higher trypanocidal activity against epimastigote forms of T. cruzi, displaying inhibitory concentration (IC₅₀) values of 0.67, 3.89 and 31.35 μM, respectively. These compounds were also evaluated against intracellular amastigote forms of T. cruzi, with five displaying similar activity to benznidazole. In vivo assays showed significant reduction of parasitaemia after administration of five in mice infected.