Viriditoxin is one of the 'classical' secondary metabolites produced by fungi and that has antibacterial and other activities; however, the mechanism of its biosynthesis has remained unknown.Here, a gene cluster () responsible for viriditoxin synthesis was identified, via a bioinformatics analysis of the genomes of and that both are viriditoxin producers. The function of the eight-membered gene cluster of was characterized by targeted gene disruptions, revealing the roles of each gene in the synthesis of this molecule and establishing its biosynthetic pathway, which includes a Baeyer-Villiger monooxygenase catalyzed reaction. Additionally, a predicted catalytically-inactive hydrolase was identified as being required for the stereoselective biosynthesis of ()-viriditoxin. The subcellular localizations of two proteins (VdtA and VdtG) were determined by fusing these proteins to green fluorescent protein, to establish that at least two intracellular structures are involved in the compartmentalization of the synthesis steps of this metabolite.The predicted pathway for the synthesis of viriditoxin was established by a combination of genomics, bioinformatics, gene disruption and chemical analysis processes. Hence, this work reveals the basis for the synthesis of an understudied class of fungal secondary metabolites and provides a new model species for understanding the synthesis of biaryl compounds with a chiral axis.