Port Phillip Bay is located on the central south coast of Victoria, Australia. It is a large urbanised marine embayment encompassing an area of roughly 1950 km2 with a coastline approximately 264 km; and hosts a population of over four million people on its catchment basin. The Bay receives waters from the Yarra River, discharges from sewage treatment plants and agricultural inputs. It is home to a wide range of fish species, with seagrass beds used as nursery sites. The southern sand flathead (Platycephalus bassensis) are long-lived carnivorous ambush predators that have a sedentary, non-migratory lifestyle. They conceal themselves in fine sediments, are not strong swimmers and are believed to be representative of the area from where they are collected. Consequently they are considered a suitable bioindicator species for their local environment. The aim of this study is to use metabolomics to investigate responses of sand flathead to different environments in Port Phillip Bay. Two year old female fish were collected from five sites within the bay. Assessment of the fish for general condition showed differences between fish from urbanised/industrialised sites (Corio Bay, Hobsons Bay, Mordialloc) compared to fish from low population density areas (Sorrento and St Leonards). For the metabolomics analysis, livers were subjected to polar metabolite analysis and free fatty acid analysis using gas chromatography mass spectrometry. The PCA results for polar metabolites showed that the samples from Sorrento separated from those of the other sites. Notably, fish from Sorrento and Mordialloc showed significant differences in amino acids, glycolytic and TCA intermediates. Interestingly, results for the free fatty acid analysis showed that samples from Corio Bay separated from those of other sites. The present study highlights the value of using metabolomics which can offer insights into sand flathead responses to different environments within Port Phillip Bay.