Several studies have indicated the presence of contaminants in Alpine aquatic ecosystems. Even if measured concentrations are far below those that cause acute effects, continuous exposure to sub-lethal concentrations may have detrimental effects on the aquatic species present in these remote environments. This may lead to a cascade of indirect effects at higher levels of the ecological hierarchy (i.e., the community). To improve the determination of ecologically relevant risk endpoints, behavioural alterations in organisms due to pollutants are increasingly studied in ecotoxicology. In fact, behaviour links physiological function with ecological processes, and can be very sensitive to environmental stimuli and chemical exposure. This is the first study on behavioural alteration in a wild population of an Alpine species. In the present study, a video tracking system was standardized and subsequently used to identify contaminant-induced behavioural alterations in Diamesa zernyi larvae (Diptera, Chironomidae). Diamesa zernyi larvae, collected in an Italian Alpine stream (Rio Presena, Trentino Region), were acclimated for 24 h and successively exposed to several aquatic contaminants (pesticides: chlorpyrifos, metolachlor, boscalid, captan; pharmaceuticals: ibuprofen, furosemide, trimethoprim) at concentrations corresponding to their Lowest Observed Effect Concentration (LOEC). After 24, 48, 72, and 96 h of exposure, changes in the distance moved, the average speed, and the frequency of body bends were taken to reflect contaminant- and time-dependent effects on larval behaviour. In general, metolachlor, captan, and trimethoprim tended to reduce all the endpoints under consideration, whereas chlorpyrifos, boscalid, ibuprofen, and furosemide seemed to increase the distances moved by the larvae. This could be related to the different mechanisms of action of the investigated chemicals. Independently of the contaminant, after 72 h a general slowing down of all the behavioural activities occurred. Finally, we propose a behavioural stress indicator to compare the overall behavioural effects induced by the various contaminants.