BACKGROUND: Given the global rise in antimicrobial resistance, the discovery of novel antimicrobial agents and production processes thereof are of utmost importance. To this end we have activated the gene cluster encoding for the biosynthesis of the potent antifungal compound ilicicolin H in the fungus Trichoderma reesei. While the biosynthetic gene cluster (BGC) is silent under standard cultivation conditions, we achieved BGC activation by genetically overexpressing the transcription factor TriliR. RESULTS: Successful activation was confirmed by RT-qPCR, proteomic and metabolomic analyses. Metabolomic profiling upon BGC expression revealed high-yield production of ilicicolin H. To elucidate the enzymatically highly diverse functionality of this BGC, we employed a combination of overexpression and deletions of individual genes in the BGC. While we hardly observed any of the previously reported side- or shunt products associated with heterologous ilicicolin H expression, we discovered that Trichoderma reesei produces a novel member of the ilicicolin family using a metabolomic molecular networking approach. This new compound, ilicicolin K, is expressed in substantial amounts in the genetically engineered Trichoderma reesei. Ilicicolin K differs from ilicicolin H in its structure by a second hydroxylation of the tyrosine derived phenol and an additional ring formed by an intramolecular ether bridge of the hydroxyl group at the pyridone towards the tyrosine moiety of the molecule. Bioactivity tests of ilicicolin K revealed a strong antifungal activity against Saccharomyces cerevisiae and a moderate activity against the human pathogen Candida auris, an emerging multi-drug resistant fungus. CONCLUSIONS: By activating a silent BGC in T. reesei, we obtained a high-yielding strain for the production of the antifungal compounds ilicicolin H and the novel ilicicolin K. These two compounds share some structural properties and are thus highly likely to act on the fungal cytochrome bc1 complex, a component of the mitochondrial repository chain. However, they possess different bioactive properties, which might suggest that ilicicolin K may overcome certain limitations of ilicicolin H.