Harbour sediments include wash-off from different nearby catchments that have various landuse activities. In this study the spatial variation of polycyclic aromatic hydrocarbons (PAHs) in Sydney Harbour was studied by analysing the sediment. The sediment was collected from 12 locations and 16 PAHs that are prioritized by the United States Environmental Protection Authority were analysed. The concentrations in the sediment were in the range of 54–23,440 ng/g of total PAHs and this varied from location to location. The result suggests that the harbour is contaminated with PAHs from low concentration to very high concentration. Among the total PAHs, approximately 75% were medium to higher molecular weight 4- and 5-ring member PAHs. A toxicity identification evaluation (TEQ) approach using a toxicity equivalency factor (TEF) was applied to assess the sediment toxicity. The TEF was found to be between 22 and 8,277 ng TEQ/g. The TEQ value indicated potential adverse ecological and human health effects in many locations.