The introduction of marine pests to new environments is often facilitated by commercial vessels that can transport species as either hull fouling or in their ballast water. A collaborative project between Agriculture Victoria, the Port of Melbourne, Victorian Ports Corporation, Deakin University and EnviroDNA was established in 2021 to use eDNA to analyse settlement arrays, plankton tows and water samples to detect fouling species and larvae in the water column. This initial pilot was successful and ongoing monitoring will contribute to the establishment of a long-term program for all of Victoria’s high-risk commercial marine ports.