Neonicotinoid mixtures are common in streams worldwide, but corresponding ecological responses are poorly understood. We combined experimental and observational studies to narrow this knowledge gap. The mesocosm experiment determined that concentrations of the neonicotinoids imidacloprid and clothianidin (range of exposures, 0 to 11.9 μg/liter) above the hazard concentration for 5% of species (0.017 and 0.010 μg/liter, respectively) caused a loss in taxa abundance and richness, disrupted adult emergence, and altered trophodynamics, while mixtures of the two neonicotinoids caused dose-dependent synergistic effects. In 85 Coastal California streams, neonicotinoids were commonly detected [59% of samples ( n = 340), 72% of streams], frequently occurred as mixtures (56% of streams), and potential toxicity was dominated by imidacloprid (maximum = 1.92 μg/liter) and clothianidin (maximum = 2.51 μg/liter). Ecological responses in the field were consistent with the synergistic effects observed in the mesocosm experiment, indicating that neonicotinoid mixtures pose greater than expected risks to stream health.