Environmental DNA (eDNA) is increasingly used as a non-invasive tool for marine biodiversity monitoring in Australia, supporting applications including marine park surveillance, biosecurity, fisheries management, and environmental assessments for offshore developments. We applied eDNA metabarcoding to characterise fish communities during the first two voyages of the South-East Australian Marine Ecosystem Survey (SEA-MES) in June 2023 and May 2024. Seawater samples were collected aboard the RV Investigator from 91 sites along the continental shelf edge (~80–500 m depth), extending from eastern Tasmania to southern New South Wales. Across all samples, we detected 230 fish taxa, including 138 identified to species level and 77 to genus level. Multivariate analyses revealed significant differences in eDNA-derived community composition between voyages, indicating temporal variability. Fish assemblages also differed consistently between surface and near-bottom samples, with bottom-water samples detecting approximately twice as many species on average. After accounting for other sources of variation, clear spatial structuring of fish communities emerged, associated with both seafloor depth and latitude. These findings demonstrate the capacity of eDNA to resolve spatial and temporal patterns in marine fish assemblages across broad environmental gradients and highlight its value as a complementary tool for large-scale ecosystem surveys.