Sub‐lethal toxicological responses are common occurrences in aquatic animals exposed to sewage wastewater and organic wastewater contaminants. Behavioural alterations are particularly sensitive indicators of sub‐lethal toxicological stress in animals exposed to various pollutants, and often correlate with higher‐level outcomes. Diurnal activity patterns in many fish species are sensitive to changes in natural biotic factors, but few studies have explored how environmental pollutants influence such rhythms. We investigated diurnal activity patterns in the mosquitofish (Gambusia holbrooki), after exposure to UV‐treated sewage and a mixture of key contaminants identified through chemical analysis and subsequent risk‐based prioritization of the wastewater. Exposure to 50% and 100% wastewater abolished daytime activity levels in male, but not female fish. Chemical analysis identified fluoxetine, diazinon and triclosan above their reported predicted‐no‐effect‐concentrations (PNECs), and fish were thus exposed to a mixture of these compounds at 1, 10 and 100 μg l–1. Behavioural responses were highly consistent between fish exposed to wastewater and the contaminant mixture, indicating that these prioritized contaminants are indeed likely contributing to the observed effects. Effective concentrations of the mixture were considerably lower than those reported as eliciting behavioural effects in previous studies exploring each of these compounds alone. Results warn of the potential for negative higher‐level consequences associated with exposures of fish to common organic wastewater contaminants, as altered diurnal activity patterns could conceivably scale‐up to influence performance including foraging success and predator avoidance. Further research is necessary to increase our understanding of linkages between alterations to diurnal activities and effects at the population level.