Port Phillip Bay is a large, shallow semi-enclosed bay that supports diverse marine life, sustains a wide range of human activities, and provides significant benefits for the Victorian economy. The Victorian Government’s Environmental Management Plan aims to conserve and enhance the health of the marine and coastal environments, ensuring sustainable use of resources. The Department of Energy, Environment and Climate Action instigated a study to understand condition of sediment habitats and benthic communities to inform decision-making and monitoring. The Port Phillip Bay Seafloor Integrity Project involves sampling of sediments to study biological, physical, and chemical properties of sediments and associated biogeochemistry processes, as well as undertaking ecotoxicological studies and underwater imaging surveys of benthic ecosystem. Using data collected across this multidisciplinary study, we compare conditions between areas influenced by human activities and areas believed to be in good conditions, to understand the activities impact on sediments and inform managers’ decisions. We identify indicators of condition and use them to develop a Seafloor Integrity score. Indicators were selected to allow for early detection of ecological change and enable adaptive management. Selected indicators will contribute to the regular monitoring and assessment of the Bay status against targets that reflect Good Environmental Status.