The impacts of treated sewage effluent on the receiving waters of tropical estuaries are still unclear. Darwin Harbour, in the Northern Territory, is subject to various anthropogenic influences, including annual inputs of > 10,000 ML of treated sewage effluents. Water quality targets are needed to effectively measure ecosystem health but few biological indicators appropriate for tropical estuaries have been identified. Previous studies at two Darwin Harbour estuaries that receive sewage effluent, found a transient effect in the water of the less impacted, well-flushed estuary with a sediment denitrification efficiency of 90 % as compared to an efficiency of <10 % for the more impacted, poorly flushed estuary. We use a nitrogen cycle microarray developed by CSIRO Hobart to determine if changes to denitrification efficiency, and other changes in N-cycle processes are reflected in the functional genes of microbes associated with the nitrogen cycle. We sample with a single impact-double control study design, over several years, seasons and tidal states (spring, neap), and collect physico-chemical, nutrient, metal and bacterial community data of water and sediment from two sewage effluent impacted, and four reference creeks, in and around Darwin Harbour. This work has implications for routine monitoring if it can be shown that changes to functional level genes can be detected before major changes to ecosystem health are observed.