Freshwater environments are common repositories for the discharge of large volumes of domestic and industrial waste, particularly through wastewater effluent. One common group of chemical pollutants present in wastewater are endocrine disrupting chemicals (EDCs), which can induce morphological and behavioural changes in aquatic organisms. The aim of this study was to compare the reproductive behaviour and morphology of a freshwater fish, the mosquitofish (Gambusia holbrooki), collected from two sites (wastewater treatment plant (WWTP) and a putative pristine site). The mosquitofish is a sexually dimorphic livebearer with a coercive mating system. Males inseminate females using their modified anal fin as an intromittent organ. Despite this, females are able to exert some control over the success of male mating attempts by selectively associating with, or avoiding, certain males over others. Using standard laboratory assays of reproductive behaviour, we found that mosquitofish males living in close proximity to WWTP showed increased mating activity compared to those inhabiting a pristine site. More specifically, during behavioural trials in which males were allowed to interact with females separated by a transparent divider, we found that WWTP-males spent more time associating with females. Concordant with this, when males and females were subsequently allowed to interact freely, WWTP-males also spent more time chasing and orienting towards the females. As a result, females from both sites showed more interest towards the WWTP-site males. Male anal fin morphology, however, did not differ between sites. Our study illustrates that lifetime exposure to WWTP-effluents can greatly affect male behaviour. The results underscore the importance of behaviour as a potential tool for investigating unknown contaminants in the environment.