Constructed wetlands are popular tools for managing threatened flora and fauna in urban settings, but there are concerns that these habitats may increase mosquito populations and mosquito-related public health risks. Understanding the interactions occurring between mosquitoes of public health concern and co-occurring organisms is critical to informing management of these habitats to mitigate potential health risks and balance the multiple values of urban wetlands. This study examined how oviposition behavior of Culex annulirostris Skuse, the most important pest mosquito species associated with freshwater wetland habitats in Australia, is influenced by the presence of Gambusia holbrooki Girard, a widespread invasive fish. Water was collected from urban wetlands that are intensively managed to reduce G. holbrooki populations to assist conservation of locally threatened frogs, and adjacent unmanaged wetlands where G. holbrooki was abundant. Laboratory experiments were conducted to examine the oviposition response by Cx. annulirostris to water samples from these two habitats. Experiments were conducted on two occasions, once in February following draining and refilling of the urban wetlands, and repeated following a substantial rainfall event in March. The results clearly demonstrate that ovipositing mosquitoes were able to detect and avoid water derived from habitats containing fish, even in the absence of the fish themselves. Understanding how invasive species affect the behavior and spatial distribution of pest species such as Cx. annulirostris will enable future wetland design and management to maximize benefits of urban wetlands and minimize potential public health risks.