Antimicrobial metabolites produced by Trichoderma koningii SMF2 exhibited antimicrobial activity against a range of Gram-positive bacterial and fungal phytopathogens. Purification of these metabolites was achieved using combinations of gel filtration and high-performance liquid chromatography. Identified by liquid chromatography electrospray ionization tandem mass spectrometry, the active metabolites proved to be three known peptaibols: Trichokonin VI, VII and VIII. The Trichokonins were stable and remained biological active over a wide pH range and at every temperature tested, showing no loss of activity even after autoclaving. Trichokonins were insensitive to proteolytic enzymes. Trichokonin VI takes on typical helical structure and the structure changes only slightly at different temperatures and pH values. The present study presented the potential of Trichokonins to be used as biological control agents.