Neonicotinoids are widely used pesticides whose harmful effects on biodiversity have been long recognized. This work analyzes the incidence and prevalence of five neonicotinoids in four different taxa of aquatic invertebrates in three sites of the Seyon River, in Switzerland. All samples analyzed were contaminated with at least one neonicotinoid, and two neonicotinoids found in the samples had been banned from use nine months prior to sampling: these facts highlight the substances’ ubiquity and high persistence in natural environments. The concentrations indicated a chronic exposure to neonicotinoids, except for one value which was ten times bigger than the others. These alarming results encourage further studies on the subject, in which the experimental methods developed for this work could be exploited.