Fishes represent an important natural resource and yet their diversity and function in dynamic estuaries with relatively high levels of human pressure such as Sydney Harbour have rarely been quantified. Further, Eastern Australia supports the survival and persistence of an increasing number of tropical species found within temperate estuaries owing to increasing average ocean temperatures. A re-valuation of the number of fish species known from Sydney Harbour is therefore needed. In this study, we generated an up-to-date and annotated checklist of fishes recorded from Sydney Harbour based on verified natural history records as well as newly available citizen science records based on opportunistic observations and structured surveys. We explored the spatial and temporal distribution of these records. In addition, we quantified the function, conservation status, and commercial importance of the identified fishes. The number of fish species recorded from Sydney Harbour now stands at 675, an increase of 89 species (15 %) when compared to the most recent evaluation in 2013. We attribute this increase in fish diversity over a relatively short time to the contribution of newer citizen science programs as well as the influx and survival of fishes in the Harbour with preferences for warmer waters. Some fish families were also overrepresented in the more urbanized and polluted sections of the Harbour. In forecasting further environmental impacts on the fishes of Sydney Harbour, we recommend increased integration of collaborative citizen science programs and natural history collections as a means to track these changes.