ome of the most powerful natural antimicrobial compounds originate from filamentous fungi. However, due to the diversity of compounds from plant and fungal origin, separation, isolation, and identification of bioactive constituents can be a long and tedious process. This study explores the effectiveness of thin layer chromatography (TLC) in combination with bioautography in the separation and identification of bioactive compounds from several filamentous fungi. Ultra-performance liquid chromatography coupled to photodiode array detector (UPLC-DAD) was employed to quantitatively identify phenolic composition. The total phenolic content of the selected filamentous fungi ranged from 31.85 mg g−1 to 101.77 mg g−1. Additionally, liquid chromatography mass spectrometry (LC/MS) determined the most abundant fatty acids were linoleic, palmitic, oleic, and stearic acid. Submerged cultivation of Grifola frondosa, Monascus purpureus, Lentinula edodes, Trametes versicolor and Pleurotus ostreatus proved to be an effective method to produce natural antimicrobial compounds.