Trichoderma brevicompactum, T. viride, T. harzianum, T. atroviride, T. longibrachiatum, T. erinaceum, T. citrinoviride, and Hypocrea lutea were screened for production of trichothecenes after growth on one or several solid and liquid media. Trichothecenes were detected by liquid chromatography combined with online UV/vis spectroscopy and electrospray high-resolution mass spectrometry. T. brevicompactum produced trichodermin and/or harzianum A on all media investigated, with liquid media yielding the largest amounts. Detection of octa-2Z,4E,6E-trienedioic acid in the harzianum-A-producing strains indicated that harzianum A was synthesized directly by esterification of trichodermol with octa-2Z,4E,6E-trienedioic acid. Both the T. viride strain from which trichodermin was originally isolated and the T. harzianum strain from which harzianum A was originally isolated were shown to belong to T. brevicompactum based on four independent criteria: metabolite profiles, micromorphology, macromorphology on yeast extract sucrose agar and potato dextrose agar, and DNA sequences of the ITS1/ITS2 regions of the nuclear ribosomal DNA.