Abstract Objective This work describes the isolation of anti-Leishmania amazonensis metabolites from Saururus cernuus (Saururaceae). Additionally, ultrastructural changes in promastigotes were evidenced by electron microscopy. Methods The MeOH extract from the leaves of S. cernuus was subjected to bioactivity-guided fractionation. Anti-L. amazonensis activity of purified compounds was performed in vitro against promastigote and amastigote forms. Key findings Bioactivity-guided fractionation of the MeOH extract from the leaves of S. cernuus afforded two related tetrahydrofuran dineolignans: threo,threo-manassantin A (1) and threo,erythro-manassantin A (2). Compounds 1 and 2 displayed activity against promastigotes (EC50 of 35.4 ± 7.7 and 17.6 ± 4.2 μm, respectively) and amastigotes (EC50 of 20.4 ± 1.9 and 16.0 ± 1.1 μm, respectively), superior to that determined for the positive control miltefosine (EC50 of 28.7 ± 3.5 μm). Reduced cytotoxicity for host cells was observed for both compounds. Additionally, ultrastructural changes in promastigotes leading to an alteration of structural morphology were observed, as evidenced by electron microscopy. Furthermore, these compounds altered the morphology and physiology of the plasmatic membrane of L. amazonensis. Conclusions The obtained results indicated that dineolignans 1 and 2 could be considered as a scaffold for the design of novel and selective drug candidates for the treatment of leishmaniasis.