ABSTRACT Increased temperatures and exposure to agricultural insecticides are increasingly threatening freshwater ecosystems worldwide. However, their combined effects are still poorly understood. We investigated the individual and interactive effects of water temperature and imidacloprid, the most widely used neonicotinoid insecticide, on larvae of two New Zealand mayflies, Deleatidium spp. and Coloburiscus humeralis. Ninety-six-hour bioassays with imidacloprid were performed at 9, 12, 15, 18, 21 and 24°C, in a full-factorial design. Mayfly survivorship and sub-lethal responses of impairment and moulting were recorded. We documented synergistically increased mayfly mortality and immobility with exposure to imidacloprid at higher temperatures, implying temperature-enhanced toxicity of imidacloprid. Survivorship of both mayflies was synergistically reduced by the combination of imidacloprid exposure and increasing temperatures. The same interaction also affected mobility of C. humeralis and moulting frequency of Deleatidium. Our findings suggest that developing a more thorough understanding of temperature-contaminant interactions may be important to allow better protection of aquatic ecosystems.