The extensive utilization of antibiotics and other pharmaceuticals has led to a concerning transformation of polluted aquatic ecosystems globally. Among these antibiotics, there is an increasing detection of erythromycin, a representative of macrolides, which induces toxic effects on non-target aquatic organisms. The ecotoxicity of erythromycin in freshwater was assessed using Cyprinus carpio during a 96-hour acute exposure. Toxicity was evaluated using a sublethal concentration of 2.5 mg/L of erythromycin, with a focus on behavioural endpoints, as behavioural biomarkers are non-invasive, ecologically relevant, detectable at low toxic concentrations, and provide early warning signals regarding the health of the exposed population. Behavioural responses were monitored at 24, 48, 72, and 96 hours. The observed behavioural alterations included hyperactivity, increased operculum movement and swimming rate, dark pigmentation, excessive mucus secretions, loss of equilibrium, surface air gulping, aggregation, lethargy, caudal bending, responsiveness to stimuli or sensitivity, and shedding of scales. These behavioural responses may indicate respiratory impairment, neurotoxic effects, and defensive responses to minimize the irritation caused by erythromycin. Consequently, the findings suggest that behavioural biomarkers serve as a crucial tool for observing and evaluating environmental health conditions. There is an urgent need to monitor erythromycin concentration and its toxicity in aquatic ecosystems.