Trichosetin, a tetramic acid-containing metabolite produced in the dual culture of Trichoderma harzianum and Catharanthus roseus (L.) G. Don callus, was subjected to phytotoxicity assays. In seedling growth assays, trichosetin inhibited root and shoot growth of all five plant species tested by damaging the cell membrane, as evidenced by the dose-dependent increase in electrolyte leakage and lipid peroxidation. Vital staining of trichosetin-treated Nicotiana tabacum BY-2 cells, with rhodamine 123, showed a weaker green fluorescence compared to controls indicating damaging effects on mitochondria. FDA-PI staining, to determine cell viability, indicated that cells of the trichosetin-treated roots were mostly dead.