The genus Coccoloba (Polygonaceae, order Caryophylalles) comprises approximately 150 species of flowering plants. Coccoloba cowellii Britton is an endemic and critically endangered representative of this genus that only grows in the municipality of Camagüey, province of Cuba. Preliminary investigation of its total methanolic extract led to the discovery of promising antifungal activity. In the present study, a bioassay-guided fractionation by means of flash chromatography and employing 1D and 2D NMR, and NMR-based machine learning tool “Small Molecule Accurate Recognition Technology” (SMART 2.1, available at https://smart.ucsd.edu/classic), allowed the isolation of quercetin and four methoxyflavonoids: 3-O-methylquercetin, myricetin 3,3',4'-trimethyl ether, 6-methoxymyricetin 3,4'-dimethyl ether, and 6-methoxymyricetin 3,3',4'-trimethyl ether. The leaf extract, fractions, and compounds were tested against various fungi and showed a strong antifungal effect against Cryptococcus neoformans and various Candida spp. with no cytotoxicity (CC₅₀ > 64.0 µg/mL) on human foetal lung fibroblasts (MRC-5 SV2 cells), determined by the resazurin assay. The total methanolic extract showed higher and broader activity when compared with the fractions and mixture of compounds.