The brasiliquinones are a family of benz[a]anthraquinone antibiotics with activity against Gram-positive and acid-fast bacteria. Despite their discovery over two decades ago, the biosynthetic locus responsible for production of the brasiliquinones has remained hidden. Here, we used a combined genomic and genetic approach to identify and clone the brasiliquinone biosynthetic gene cluster from a human-pathogenic isolate of Nocardia brasiliensis using transformation-associated recombination (TAR) in yeast. We show that this locus is conserved in other human-pathogenic N. brasiliensis and Nocardia vulneris genomes and that it is silent in these organisms, but activated upon heterologous expression in a Streptomyces host. This work provides the basis for linking quiescent biosynthetic loci to their cognate metabolites in the genus Nocardia and forms the first step in rational engineering attempts on the brasiliquinone pathway.