Port Phillip Bay is highly valued as an environmental asset and is used by a large population of stakeholders in numerous categories. Owing to a variety of activities in the marine environment and its catchments, including urbanisation, industry, shipping, aquaculture, and agriculture, the Bay is subject to multiple pressures which could present risks to environmental values. Despite these facts, there is a paucity of available data on organic and metal contamination in the Bay, and monitoring has been limited. Fathom Pacific led a Department of Energy, Environment, and Climate Action funded study, the Seafloor Integrity Project, which assessed contamination in sediments and five animal species across the Bay. The study aimed to identify indicators of seafloor condition and to provide baseline data for future monitoring. We found evidence of sediment contamination and bioaccumulation in fauna, including organics in sediments, and mercury in a popular recreational fishing species. Regional trends could be identified as well as spatial associations of contamination with specific activities. Several of our findings suggest that there are current ecological risks, including risks to recreational fishing and aquaculture in some areas of the Bay. We provided recommendations based on our findings.