The development of new antibiotics is key to addressing the crisis in human health caused by the rise of multi-drug resistant superbugs. Empirical screening of bacteria and fungi for bioactive molecules has been the source of the most successful existing antibiotics. The most prolific producers of these metabolites are the Actinobacteria, particularly the genus Streptomyces, but high re-discovery rates amongst soil-derived organisms demand the testing of new reservoirs of biodiversity and bioactive molecules. Recent studies have shown that human-associated bacteria represent a previously untapped source of antimicrobial diversity. Here, we describe our experiences exploring the antimicrobial activity of a diverse culture collection of 700 human pathogenic Actinobacteria held by our state microbiology reference laboratory. We show that organisms from this collection produce compounds capable of inhibiting the growth of the hospital superbugs MRSA and VRE. To investigate the genetics behind the production of these antibiotics, we have sequenced 100 of these genomes, which has provided a glimpse into the immense secondary metabolic potential of these organisms. Furthermore, we have cloned and expressed several large gene clusters for the biosynthesis of predicted antibiotics in heterologous hosts, which is the first time this has been achieved using Nocardia species. Our investigation of this collection of strains, via a combination of molecular biology, genomics and chemical biology, paves the way for further discoveries capable of refilling the antibiotic pipeline.