β-adrenergic receptors (βARs) belong to a key molecular targets that regulate the most important processes occurring in the human organism. Although over the last decades a zebrafish model has been developed as a model complementary to rodents in biomedical research, the role of β₂AR in regulation of pathological and toxicological effects remains to elucidate. Therefore, the study aimed to clarify the role of β₂AR with a particular emphasis on the distinct role of subtypes A and B of zebrafish β₂AR. As model compounds selective β₂AR agonists – (R,R)-fenoterol ((R,R)-Fen) and its new derivatives: (R,R)-4’-methoxyfenoterol ((R,R)-MFen) and (R,R)-4'-methoxy-1-naphtylfenoterol ((R,R)-MNFen) – were tested. We described dose-dependent changes observed after fenoterols exposure in terms of general toxicity, cardiotoxicity and neurobehavioural responses. Subsequently, to better characterise the role of β₂-adrenergic stimulation in zebrafish, we have performed a series of molecular docking simulations. Our results indicate that (R,R)-Fen displays the highest affinity for subtype A of zebrafish β₂AR and β_2AAR might be involved in pigment depletion. (R,R)-MFen shows the lowest affinity for zebrafish β₂ARs out of the tested fenoterols and this might be associated with its cardiotoxic and anxiogenic effects. (R,R)-MNFen displays the highest affinity for subtype B of zebrafish β₂AR and modulation of this receptor might be associated with the development of malformations, increases locomotor activity and induces a negative chronotropic effect. Taken together, the presented data offer insights into the functional responses of the zebrafish β₂ARs confirming their intraspecies conservation, and support the translation of the zebrafish model in pharmacological and toxicological research.