Isoquinoline alkaloids, especially from the Annonaceae family, have shown biological potential against parasites. Thus, this study aimed to evaluate the potential of the alkaloid fractions of the plant Fusaea longifolia against Plasmodium falciparum and annotate the compounds present in these samples. The tentative characterization of the alkaloids from the leaves and branches of F. longifolia was performed using liquid chromatography coupled to mass spectrometry (LC-MS/MS) and molecular networks. Through manual interpretation of the MS/MS spectra, 18 alkaloids were dereplicated from F. longifolia, 17 of which were reported for the first time in this species. An unpublished putative glycosylated alkaloid was annotated by interpreting the fragmentation data profile. Regarding biological activity, the fractions studied showed high activity against P. falciparum with half-maximal inhibitory concentration (IC50) of 2.42 and 1.60 μg mL-1 for branches and from the leaves, respectively, both similar to the reference standard quinine (IC50 of 1.24 μg mL-1). The structures of the 17 alkaloids were subjected to in silico analysis using molecular docking against four enzymes related to anti-Plasmodium activity (wild type (dm-PfDHFR) and mutant type (qm-PfDHFR), dihydroorotate dehydrogenase (PfDHODH) and purine nucleoside phosphorylase (PfPNP)). Molecular docking revealed strong interactions, especially between oxoxylopine 17 and hydroxycassythicine N-oxide 10, which may be potential new sources against P. falciparum.