Neonicotinoid insecticides are widely used agents in agriculture to control a broad range of insect pests. Although use of neonicotinoid pesticides has resulted in the widespread contamination of surface waters, sublethal toxicity data of these products in relation to non-target aquatic biota are still poor. Therefore, the objective of this study was to assess the effects of two neonicotinoid pesticides with widespread use on the basic physiological functions: the thoracic limb activity and heart rate of Daphnia magna, and to screen for their potential to affect the cytochrome P450 monooxygenase system of daphnids. The considered pesticides were the acetamiprid- and thiacloprid based products Mospilan 20 SG and Calypso 480 SC. The dose-dependent variation in the three biological endpoints considered were assessed following 24h exposures. The two neonicotinoid formulations elicited significant depression on the thoracic limb activity and heart rate of daphnids at doses close to the 48h-EC50 of the products, a response mainly attributable to the overall drop in the general health status of the organisms. The dose related variation in the ECOD activity of daphnids exposed to the selected neonicotinoid formulations followed a biphasic pattern, with starting effective doses for Mospilan 20 SG of 6.3 mg L-1 (= 1/20 of 48h-EC50 for Daphnia neonates), and for Calypso 480 SC of 0.034 mg L-1 (= 1/4000 of 48h-EC50). Maximal ECOD activity (2.2 fold increase vs. controls) was induced by Mospilan 20 SG in daphnids exposed to 114 mg L-1 product (= 48h-EC20), and by Calypso 480 SC (1.8 fold increase) at 5.2 mg L-1 dose (= 1/20 of 48h-EC50). The results outlined significant alterations in the physiological traits considered at concentrations below the immobility thresholds (48h-EC50) of the products used as benchmarks to rate their toxicity risks to aquatic biota. Therefore, we think our findings might deserve consideration in the environmental risk evaluation of these products.