Procaine penicillin (PP) is a β-lactam antibiotic widely used in human and veterinary medicine. Although PP is detected in surface water, little is known on its effects on aquatic invertebrates. Our aim was to determine the influence of PP on swimming behaviour (track density, swimming speed, turning angle, hopping frequency) and physiological activity (oxygen consumption, heart rate, thoracic limb movement) of a freshwater invertebrate Daphnia magna exposed to PP at concentrations of 11.79 mg/L, 117.9 mg/L and 1179 mg/L for 2 h and 24 h. The results showed no mortality; however, reduction of swimming activity manifested by the decreased track density, swimming speed and turning angle noted in Daphnia exposed to all the concentrations of PP. Increase of oxygen consumption was observed after 2-h exposure; however, decrease of this parameter was found after 24 h. PP also reduced heart rate and thoracic limb movement in a concentration-dependent manner. The results suggest that the antibiotic should not induce mortality; however, it may affect swimming behaviour and physiological parameters of Daphnia magna particularly inhabiting aquaculture facilities with intensive antibiotic treatment. On the basis of the present results, we also suggest higher sensitivity of behavioural and physiological parameters of cladocerans than the commonly used endpoints: mortality or immobilisation and their possible application as a part of early warning systems in monitoring of surface water toxicity.