Clusia grandiflora Splitg. (Clusiaceae), popularly known as “cebolão” or “orelha-de-onça”, is a Neotropical species recognized as a rich source of bioactive secondary metabolites, particularly prenylated benzophenones. This study investigated the chemical composition and antileishmanial potential of the ethanolic extract (EBCG), dichloromethane fraction (FDCG), and hexane fraction (FHex) obtained from C. grandiflora fruits. Chemical profiling by HPLC-ESI-IT-MS/MS revealed a metabolome dominated by prenylated benzophenones, terpenes, and flavonols. Biological evaluation included activity against promastigote and axenic amastigote forms of Leishmania (L.) amazonensis, cytotoxicity toward RAW 264.7 macrophages, assessment of cell death phenotypes, nitric oxide (NO) production, and molecular docking against Leishmania infantum trypanothione reductase (TRLi). FDCG and FHex exhibited moderate activity against promastigotes (IC₅₀ = 20.34 and 85.86 µg mL⁻¹, respectively), whereas EBCG was inactive. FHex showed the highest activity against axenic amastigotes (IC₅₀ = 7.0 µg mL⁻¹), while EBCG and FDCG displayed moderate activity (IC₅₀ = 15.92 and 26.43 µg mL⁻¹, respectively). Cell death occurred predominantly through a necrosis-like phenotype in promastigotes and an apoptosis-like phenotype in axenic amastigotes, and all samples significantly reduced NO production. Molecular docking indicated that several putatively annotated metabolites from C. grandiflora can be favorably accommodated within the TS2 substrate-binding cavity of Leishmania infantum trypanothione reductase (TRLi). Garcinielliptone I and Guttiferone E exhibited the most favorable predicted binding affinities, whereas xanthocymol displayed an interaction pattern involving the catalytic residues Cys52 and Cys57. These findings highlight selected putatively annotated metabolites as promising candidates for future biochemical investigation. Overall, these findings highlight C. grandiflora fruits as a promising, previously underexplored source of prenylated benzophenones with antileishmanial activity and the ability to reduce nitric oxide production in LPS-stimulated RAW 264.7 macrophages.