Formulations of spinosad for mosquito and midge control (e.g., Natular®) are derived from the Saccharopolyspora spinosa bacterium. They offer a different mode of action from other larvicides. We tested the effectiveness of single-brood (2EC) and extended-release pellet (G30) formulations against larvae of Culex tarsalis and chironomid midges, at mid- to high label rates, in wetland mesocosms. We also monitored survival of mayfly nymphs and other nontarget insects. Both formulations were effective against mosquitoes for >4 wk and yielded excellent midge control. Spinosad caused mortality of mayflies and other nontarget insects. Spinosad was less toxic to mayflies than to targets, and effects on mayflies were undetectable after day 21. The higher toxicity for mosquitoes indicates that future research could identify rates that reduce nontarget effects while maintaining high efficacy.