Effective conservation of freshwater biodiversity depends on reliable and accurate field sampling methods for assessing species communities and key environmental assets and threats. Current techniques are generally inefficient or expensive, and often highly invasive. Environmental DNA (eDNA) has been shown to be a cost effective and sensitive method for determining species presence within freshwater environments. Recent technological advances have enabled entire communities within a waterway to be accurately identified through a process known as eDNA metabarcoding. Through a Melbourne Water initiated program, we undertook a large scale eDNA metabarcoding project to map freshwater biodiversity at 340 sites across Melbourne’s five catchments. Water sampling was undertaken over a 6-8 week period during winter, when water levels in most waterways were likely to be high. Sites included major rivers within each catchment, their tributaries, wetlands, lakes and reservoirs. We identified different animal species represented in water samples with multiple targeted metabarcoding assays. A universal vertebrate assay gave a broad overview of animal biodiversity, while three more specific assays focussed on groups of particular interest (fish, amphibians, decapods) with the aim of increasing detection sensitivity. We discuss the results of the eDNA metabarcoding surveys across each of these groups, highlighting the power of this approach for biodiversity assessments. We also contrast the results with an extensive regional database for fish and crustacean catch surveys, which date back to the early 1900s. Finally, we discuss the importance of accurate reference species DNA databases to be able to confidently assign species through the metabarcoding approach.