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. Using an experimental approach that combines the power of microbial genomics, molecular and chemical biology, we have begun exploring the antimicrobial activity of a diverse culture collection of 700 human pathogenic Actinobacteria held by our state microbiology reference laboratory. Among a random sample of 100 different species from 15 different genera we found several isolates that produce antibiotic compounds, including one novel molecule from an undescribed Streptomyces species that significantly inhibits the growth of hospital superbugs MRSA and VRE. Furthermore, sequencing of 100 of these genomes, including the complete assembly of a 9.3 Mb Nocardia strain, suggests that we have a source of considerable untapped microbial biodiversity that is rich in biosynthetic potential and will allow the identification of novel antimicrobial compounds with activity against the most troubling resistant pathogens that will help boost the antimicrobial drug discovery pipeline.