Australia’s Commonwealth Marine Park network spans diverse habitats but is dominated by deep-water environments, with more than 80% of its area deeper than 1000 m. Initial bioregionalisation was based on benthic community structure derived from trawl and video surveys, which remain central to understanding these ecosystems. However, biodiversity characterisation across such vast and remote areas remains challenging and requires additional innovative approaches. We present results from an environmental DNA (eDNA) survey conducted during a 2025 RV Investigator voyage as part of a biodiversity assessment of the southern Coral Sea Marine Park. A total of 320 eDNA samples (10 L each) were collected from 40 sites, including surface and bottom waters, across continental shelf and seamount habitats at depths of 60–4000 m. Using three fish-targeted metabarcoding assays, we detected over 500 species and observed a decline in taxonomic richness with increasing depth in near-bottom eDNA samples. Species accumulation curves indicate that detecting rare taxa requires multiple large-volume samples. Comparisons with trawl data show overlap but each survey method highlights distinct fish assemblages. These results highlight the power of eDNA to expand and refine insights from conventional Marine Park surveys.