this work aimed to determine the efficacy of the essential oil of a. gratissima as anesthetic for silver catfish, and to perform the bio-guided fractionation of essential oil aiming to isolate compounds responsible for the noted effects. fish were submitted to anesthesia bath with essential oil, its fractions and isolated compounds to determine time of anesthetic induction and recovery. eugenol (50 mg l(-1)) was used as positive control. essential oil of a. gratissima was effective as an anesthetic at concentrations of 300 to 900 mg l(-1). fish presented involuntary muscle contractions during induction and recovery. the bio-guided fractionation of essential oil furnished e-(-)-pinocamphone, (-)-caryophyllene oxide, (-)-guaiol and (+)-spathulenol. e-(-)-pinocamphone caused the same side effects observed for essential oil. (-)-caryophyllene oxide, (-)-guaiol and (+)-spathulenol showed only sedative effects at proportional concentrations to those of the constituents in essential oil. (+)-spathulenol (51.2 mg l(-1)) promoted deep anesthesia without side effects. a higher concentration of (+)-spathulenol, and lower or absent amounts ofe-(-)-pinocamphone could contribute to increase the activity and safety of the essential oil of a. gratissima. (+)-spathulenol showed potent sedative and anesthetic activities in silver catfish, and could be considered as a viable compound for the development of a new anesthetic.