Port Phillip Bay, Australia, is a large semi-closed bay with over four million people living in its catchment basin. Inflow into the Bay includes urban runoff, discharges of sewage treatment plants as well as petrochemical and agricultural chemicals. A 1999 study demonstrated that fish inhabiting the Bay showed signs of effects related to pollutant exposure, despite pollution management practices having been implemented for over a decade. To assess the current health status of the fish inhabiting the Bay, a follow up survey was conducted in 2015. A suite of biomarkers were measured to determine the health status of Port Phillip Bay sand flathead (Platycephalus bassensis); namely ethoxyresorufin-O-deethylase (EROD) activity, polycyclic aromatic hydrocarbons (PAH) biliary metabolites, carboxylesterase activity (CbE) and DNA damage (8-oxo-dG). The reduction in EROD activity in the present study suggests a decline in the presence of EROD activity-inducing chemicals within the Bay since the 1990s. Fish collected in the most industrialised/urbanised sites did not display higher PAH metabolite levels than those in less developed areas of the Bay. Ratios of PAH biliary metabolite types indicated fish collected at Corio Bay and Hobsons Bay were subjected to increased low molecular weight hydrocarbons of petrogenic origin when compared to PAH biliary metabolites in fish from Geelong Arm and Mordialloc. Quantification of DNA damage indicated a localised effect of exposure to pollutants, with a 10-fold higher DNA damage level in fish sampled from the industrial site of Corio Bay relative to the less developed site of Sorrento. Overall, integration of biomarkers by multivariate analysis indicated that the health of fish collected in industrialised areas was compromised, with biologically significant biomarkers of effects (LSI, CF and DNA damage) discriminating between individuals collected in industrialised areas from observations made in fish collected in less developed areas of the Bay.