Pest resistance against fungicides is a widespread and increasing problem, with impact on crop production and public health, making the development of new fungicides an urgent need. Chemical analyses of a crude methanol extract (CME) ofGuiera senegalensisleaves revealed the presence of sugars, phospholipids, phytosterols, guieranone A, porphyrin-containing compounds, and phenolics. To connect chemical composition with biological effects, solid-phase extraction was used to discard water-soluble compounds with low affinity for the C18 matrix and obtain an ethyl acetate fraction (EAF) that concentrates guieranone A and chlorophylls, and a methanol fraction (MF) dominated by phenolics. While the CME and MF exhibited poor antifungal activity againstAspergillus fumigatus,Fusarium oxysporumandColletotrichum gloeosporioides, the EAF demonstrated antifungal activity against these filamentous fungi, particularly againstC. gloeosporioides. Studies with yeasts revealed that the EAF has strong effectiveness againstSaccharomyces cerevisiae,Cryptococcus neoformansandCandida kruseiwith MICs of 8, 8 and 16 μg/mL, respectively. A combination of in vivo and in vitro studies shows that the EAF can function as a mitochondrial toxin, compromising complexes I and II activities, and as a strong inhibitor of fungal tyrosinase (Ki = 14.40 ± 4.49 µg/mL). Thus, EAF appears to be a promising candidate for the development of new multi-target fungicides.