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Calea uniflora Less., commonly known in Brazil as “erva-de-lagarto” and “arnica-da-praia”, is used in folk medicine to treat internal and external bruises and as anti-inflammatory. However, despite the popular use there is a lack of information in terms of isolation and biological evaluation of its secondary metabolites. Thus, the aims of this study were to identify and characterize compounds of C. uniflora leaves with trypanocidal and leishmanicidal potential. Methods: Structures of compounds were determined by HRESIMS spectra, and 1D and 2D NMR spectra. Then, they were tested for in vitro leishmanicidal and trypanocidal activities against Leishmania amazonensis and Trypanosoma cruzi intracellular amastigotes, respectively. Results: Four germacranolide-type sesquiterpene lactones were isolated and characterized from dichloromethane fraction: 2α-hydroxy-8β-2’,3’,5’-trihydroxy-angeloyloxycostunolide (1), desacetyleupaserrin (2), 2α-hydroxy-8β-3’-hydroxy-2’,5’-epoxyangeloyloxycostunolide (3) and ovatifolin (4). All compounds are described herein for the first time for C. uniflora and Calea genus. Lactones 1, 2 and 4 demonstrated weak or no inhibition of the T. cruzi amastigotes, whereas compound 3 revealed a moderate effect (36.8%). Regarding the leishmanicidal activity, 4 exhibited a 59.9% inhibitory effect on the growth of L. amazonensis. Conclusion: None of the isolated compounds presented promising trypanocidal effect and 4 was the most active compound against L. amazonensis amastigotes.