The prevalence of anxiety is a significant public health problem, being the 24th leading cause of disability in individuals affected by this disorder. In this context, chalcones, a flavonoid subclass obtained from natural or synthetic sources, interact with central nervous system (CNS) receptors at the same binding site as benzodiazepines, the primary drugs used in the treatment of anxiety. Thus, our study investigates the anxiolytic effect of synthetic chalcones derived from the natural product 2-hydroxy-3,4,6-trimethoxyacetophenone isolated from Croton anisodontus Müll.Arg. in modulating anxiolytic activity via GABAergic and serotoninergic neurotransmission in an adult zebrafish model. Chalcones 1 and 2 were non-toxic to adult zebrafish and showed anxiolytic activity via GABA_A receptors. Chalcone 2 also had its anxiolytic action reversed by the antagonist granisetron, indicating the participation of serotonergic receptors 5HTR_3A/3B in the anxiolytic effect. In addition, molecular docking results showed that chalcones have a higher affinity for the GABA_A receptor than DZP and binding in the same region of the DZP binding site, indicating a similar effect to the drug. Furthermore, the interaction of chalcones with GABA_A and 5-HT_3A receptors demonstrates the anxiolytic effect potential of these molecules. Communicated by Ramaswamy H. Sarma