Reliable, suitable DNA reference data are a fundamental requirement for the success of eDNA metabarcoding analyses and molecular species identifications. The National Biodiversity DNA Library (NBDL) is an initiative led by Australia's National Science Agency, CSIRO, to provide a reference library to support the accurate taxonomic assignment of eDNA derived from Australian ecosystems. With our partners, the NBDL is generating comprehensive DNA reference sequences from expertly identified specimens held in research collections. Our first complement of campaigns includes Australian marine vertebrates, marine invertebrates, macroalgae and seagrasses, marine pests, terrestrial vertebrates and plant pests. CSIRO has developed a cost-effective highly miniaturised genome-skimming technology ideally suited to collection specimens to build this library, in concert with efforts to host existing data generated by other initiatives that meet the same criteria. These data will be made publicly available through a dedicated data and analysis portal, with supporting specimen metadata and images. The NBDL will support the uptake of eDNA in Australian settings for community composition analyses and broader applications of molecular diagnostics, including the early detection of marine pests.