Rapid identification of known compounds, i.e., dereplication, has become a strategically important area for the natural-product chemists involved in bioprospecting of microbes for novel bioactive metabolites. Among microbial biodiversity, endophytic fungi represent an abundant and dependable source of structurally diverse bioactive metabolites. During the course of screening for antimicrobial secondary metabolites from endophytic fungi, an antimicrobial metabolite was identified from the ethyl acetate extract obtained from the culture broth of Xylaria sp., an endophytic fungus from Ficus pumila Linn. (Moraceae) that exhibited a broad spectrum of antimicrobial activity against human and phytopathogenic bacteria and fungi. Chemical investigation of the ethyl acetate fraction using thin-layer chromatography (TLC) bioautography and LC-hyphenated techniques led to the identification of a known benzoic acid derivative. Here, we describe the application of analytical strategies (TLC-bioautography) and hyphenated spectroscopic techniques (liquid chromatography-photodiode array detector-mass spectrometry [LC-PDA-MS]) for the dereplication of antimicrobial metabolites.