| Summary: | 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) of <i>Guiera senegalensis</i> leaves 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 against <i>Aspergillus fumigatus</i>, <i>Fusarium oxysporum</i> and <i>Colletotrichum gloeosporioides</i>, the EAF demonstrated antifungal activity against these filamentous fungi, particularly against <i>C. gloeosporioides</i>. Studies with yeasts revealed that the EAF has strong effectiveness against <i>Saccharomyces cerevisiae</i>, <i>Cryptococcus neoformans</i> and <i>Candida krusei</i> with 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.
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