Chagas disease, caused by Trypanosoma cruzi, remains a neglected tropical infection with limited therapeutic options and significant drug-related toxicity. Endophytic fungi are emerging as sustainable sources of bioactive metabolites with potential antiparasitic activity. In this study, endophytic fungi isolated from the medicinal plant Lippia alba were screened for trypanocidal activity. Crude extracts from seven isolates were tested against epimastigote and amastigote forms of T. cruzi, together with cytotoxicity assays in J774A.1 murine macrophages. The crude extract of Montagnula sp. exhibited the strongest trypanocidal activity (IC₅₀ = 22.1 ± 0.5 μg/mL for epimastigotes and 29.2 ± 3.0 μg/mL for amastigotes), comparable to benznidazole, with low toxicity (CC₅₀ > 600 μg/mL) and a high selectivity index (>26). Morphological and mitochondrial analyses demonstrated preservation of host-cell integrity and mitochondrial function, in contrast to the reference drug. UHPLC-ESI-HRMS profiling identified phenolic and terpenoid compounds, including caffeic acid, ferulic acid, naringenin, apigenin, and ursolic acid, which may underlie the observed biological activity. To our knowledge, this study provides the first evidence of trypanocidal activity in an endophytic Montagnula species and highlights endophytic fungi as promising platforms for the discovery of novel anti-T. cruzi agents.