The use of natural agents as anesthetics in fish management aims to minimize stress during procedures. This study aims to investigate the behavioral, electrocardiographic characteristics, and the pharmacological mechanism of action of Curcuma longa essential oil (CLEO) in Colossoma macropomum. The study was conducted on juvenile Colossoma macropomum (18.13 ± 2.1g) (n = 153), using CLEO concentrations of 125 µL.L⁻¹, 150 µL.L⁻¹, 175 µL.L⁻¹, 200 µL.L⁻¹, and 250 µL.L⁻¹, analyzing anesthetic induction and recovery behavior (Experiment I), electrocardiogram (Experiment II), and the underlying mechanism of action (Experiment III). Fish exposed to CLEO concentrations reached a deep anesthesia stage in a concentration-dependent manner. However, the effects obtained during anesthetic induction were reversible, both in behavioral parameters and cardiac activity. The mechanism of action was observed after administration of flumazenil 1 mg/kg i.p., which showed greater resistance to loss of postural reflex and shorter latency for recovery. The mechanism of action indicates involvement of inhibitory GABA neurotransmission. The cardiac effects at the concentrations used were compatible with anesthesia, but no arrhythmias occurred that could compromise the hemodynamics of the fish, demonstrating its safety for short-duration anesthesia induction in Colossoma macropomum.