SECONDARY metabolites from medicinal plants have antimicrobial properties and thus are used to cure chronic and acute diseases worldwide. This study aimed to isolate and identify the antifungal compound in the ethanolic extract of Pluchea dioscoridis leaves. Qualitative and quantitative phytochemical analysis for the secondary metabolites of P. dioscoridis was assessed. Thin layer chromatography (TLC) was used. Preliminary screening for antifungal activity against otomycotic Aspergillus niger was carried out for all isolated fractions at different concentrations. Results showed that phenolic compound RF1 exhibited excellent antifungal activity against A. niger (MIC= 2mg/mL).UV spectrum, IR, HNMR, mass spectrum and high performance liquid chromatography (HPLC) analysis identified this compound as gallic acid, with a molecular formula of C7H6O5. Therefore, gallic acid is the potential antifungal compound of P. dioscoridis extract.