An original approach was developed for the chemical and biological investigation of zone lines formed by the confrontation of fungi growing in confined spaces. Two wood-decaying fungi involved in esca disease, Eutypa lata and Botryosphaeria obtusa, were grown in Petri dishes. Metabolic profiles of pure fungal strains and confrontation zones were differentially analyzed by ultrahigh pressure liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC/TOFMS). Selected metabolites induced by the confrontation were isolated and characterized by capillary NMR (CapNMR) at the submilligram level. Fungitoxic and phytotoxic assays were applied to the crude extracts and isolated molecules. While the extracts of pure strains were inactive, the extract from confrontation zones exhibited significant activities. A very strongly induced compound, O-methylmellein, may explain these toxic properties. The developed approach demonstrates the use of fungal confrontations as an original source of bioactive molecules and gives new insights into the study of esca disease.