Pesticides with novel modes of action including neonicotinoids and anthranilic diamides are increasingly detected in global surface waters. Little is known about how these pesticides of concern interact in mixtures at environmentally relevant concentrations, a common exposure scenario in waterways impacted by pesticide pollution. We examined effects of chlorantraniliprole (CHL) and imidacloprid (IMI) on the sensitive invertebrate, Daphnia magna. Exposures were first performed using surface waters known to be contaminated by agricultural runoff. To evaluate the seasonal variation in chemical concentration and composition of surface waters, we tested surface water samples taken at two time points: during an extended dry period and after a first flush storm event. In surface waters, the concentrations of CHL, IMI, and other pesticides of concern increased after first flush, resulting in hypoactivity and dose-dependent photomotor responses. We then examined mortality and behavior following single and binary chemical mixtures of CHL and IMI. We detected inverse photomotor responses and some evidence of synergistic effects in binary mixture exposures. Taken together, this research demonstrates that CHL, IMI, and contaminated surface waters all cause abnormal swimming behavior in D. magna. Invertebrate swimming behavior is a sensitive endpoint for measuring the biological effects of environmental pesticides of concern.