The NSW Estuarine Asset Protection program (NEAP) was established in response to the devastating 2022-2023 floods on Australia’s east coast. It aimed to establish a contemporary biodiversity baseline for estuaries in New South Wales (NSW), in close collaboration with local and state government departments as well as 13 indigenous groups. The program combined environmental DNA metabarcoding surveys and sediment infauna sampling to characterise estuarine species assemblages at a state-wide scale. Since 2024, we collected 3,762 eDNA samples from 182 sites in 41 estuaries, including 12 estuaries across four marine parks. More than 12,000 taxa have been detected to date, including pest-, threatened-, and culturally significant species. Ongoing analyses are investigating anthropogenic pressures on key species groups relevant to stakeholder needs. This program aims to improve our ecological understanding of estuaries on a macroecological scale, while offering practical insights to management bodies in assessing changes in estuarine health. Through this highly collaborative program, we created the largest estuarine eDNA dataset globally, providing a comprehensive baseline of the current state of estuaries in NSW.