There is an ongoing unprecedented loss in insects, both in terms of richness and biomass. The usage of pesticides, especially neonicotinoid insecticides, has been widely suggested to be a contributor to this decline. However, the risks of neonicotinoids to natural insect populations have remained largely unknown due to lack of field-realistic experiments. Here, we used an outdoor experiment to determine effects of field-realistic concentrations of the commonly applied neonicotinoid thiacloprid on the emergence of naturally assembled aquatic insect populations. Following application, all major orders of emerging aquatic insects (Coleoptera, Diptera, Ephemeroptera, Odonata, Trichoptera) declined strongly in both abundance and biomass. At the highest concentration (10 μg/L), emergence of most orders was nearly absent. Diversity of the most species-rich family, Chironomidae, decreased by 50% at more commonly observed concentrations (1 μg/L) and was generally reduced to a single species at the highest concentration. Our experimental findings thereby showcase a causal link of neonicotinoids and the ongoing insect decline. Given the urgency of the insect decline, our results highlight the need to reconsider the mass usage of neonicotinoids to preserve freshwater insects as well as the life and services depending on them.