This dataset leverages environmental DNA (eDNA) technology to assess marine biodiversity in Australia’s offshore waters across a range of locations and depths. eDNA refers to genetic material (DNA) extracted from environmental samples such as water, soil, or air, rather than directly from organisms themselves. The eDNA in this dataset is used to identify a wide array of marine species, from microscopic plankton to larger marine life, offering a non-invasive method of biodiversity monitoring. The data has been gathered from numerous oceanographic expeditions aboard the RV Investigator over several years. These voyages span different regions of Australia's expansive offshore waters, allowing for a broad and comprehensive assessment of marine life across varying ecosystems, from shallow coastal waters to deeper, more remote offshore areas. This rich dataset includes thousands of individual water samples, collected at different sampling sites across the nation. Each sample provides genetic insights that are then analysed to build an inventory of species, revealing patterns of biodiversity that might otherwise be challenging to track with traditional surveying methods.