Introduction: Antifungal resistance is a global health problem, and alternatives for control and treatment must be sought. Thus, substances isolated from plants with antifungal capacity have received considerable attention in the pharmaceutical sector. Ouratea fieldingiana, popularly known as batiputá, is a tree species found in northeastern Brazil, with its leaves rich in biflavonoids such as amentoflavone. Biflavonoids are well-studied due to their high antifungal capacity. Objectives: Carry out the isolation, identification, and characterization of the compound amentoflavone (AMT), as well as evaluate its antifungal activity and modulatory effect against strains of Candida albicans, also including the computational study of the mechanism of action of the compound AMT against the Als3 and Sap5 enzymes of C. albicans. Methodology: Column chromatography was used to isolate the AMT compound from the species O. fieldingiana, and its characterization was performed by high-performance liquid chromatography (HPLC). The antifungal activity was analyzed by the broth microdilution method. Modulatory activity assays were performed by the checkerboard technique using AMT and amphotericin B (AmB) as a standard. For the in silico study, computational simulations of the interaction between the enzymes Als3 and Sap5, and AMT were performed using the Vina AutoDock Code. Results: The AMT compound showed an inhibitory effect against all strains of C. albicans. Regarding modulatory activity, an indifferent and antagonistic effect was observed for all C. albicans strains tested. Molecular docking with AMT showed higher affinity energy for the Als3 and Sap5 enzymes than AmB. The results obtained in this study suggest that AMT has antifungal effects, as well as a high affinity for the Als3 and Sap5 enzymes of C. albicans. Conclusion: These results prove that the AMT compound could be a potential source of a new biotechnological product, acting as a natural antifungal agent.