Sydney Harbour is a hotspot for diversity. However, as with estuaries worldwide, its diversity and functioning faces increasing threats from urbanisation. This is the first synthesis of threats and impacts in Sydney Harbour. In total 200 studies were reviewed: 109 focussed on contamination, 58 on habitat modification, 11 addressed non-indigenous species (NIS) and eight investigated fisheries. Metal concentrations in sediments and seaweeds are among the highest recorded worldwide and organic contamination can also be high. Contamination is associated with increased abundances of opportunistic species, and changes in benthic community structure. The Harbour is also heavily invaded, but invaders’ ecological and economic impacts are poorly quantified. Communities within Sydney Harbour are significantly affected by extensive physical modification, with artificial structures supporting more NIS and lower diversity than their natural equivalents. We know little about the effects of fishing on the Harbour’s ecology, and although ocean warming along Sydney is among the fastest in the world, we know little about how the ecosystem will respond to warming. The interactive and cumulative effects of stressors on ecosystem functioning and services in the Harbour are largely unknown. Sustainable management of this iconic natural system requires that knowledge gaps are addressed and translated into coherent environmental plans.