This study aimed to assess how different concentrations of the insecticide chlorpyrifos (1.1, 5.24, 11, 52.4, 110, 262, 524 and 1100 ng L) affect the swimming behaviour of Diamesa zernyi larvae following exposure. A video tracking system was employed to analyse two swimming traits (total distance moved and average speed) of the larvae simultaneously after 3 days of exposure to the pesticide at 2 °C. The behavioural results were also interpreted according to biochemical responses to oxidative stress (OS) induced by chlorpyrifos, based on malondialdehyde (MDA) and protein carbonyl (PCC) content. Both distance and speed significantly decreased after 72 h of exposure to chlorpyrifos concentrations of ≥ 110 ng L, under which significant OS was detected as lipid peroxidation (level of MDA) and protein carbonylation (level of carbonyl). Analysis of altered swimming behaviour, along with MDA and carbonyl content, indicated that ≥ 110 ng L contamination levels of the insecticide cause the organism to reallocate energy normally used for locomotor activity to repair cell damage, which might explain the strong impairment to locomotor performance. Locomotor performance is an ecologically relevant trait for elucidating the population dynamics of key species, with disturbance to this trait having long-term negative impacts on population and community structure. Therefore, chlorpyrifos insecticides represent a serious ecological risk for mountain aquatic species based on the detrimental effects observed in the current study, as the tested concentrations were those at which the insecticide is found in many Alpine rivers of Italy.