We investigated the antioxidant and cholinesterase inhibitory activities of the aqueous extract of rainbow trout Oncorhynchus mykiss. The antioxidant activity of O. mykiss aqueous extract was determined by in vitro peroxynitrite scavenging activity and reducing power assays. The aqueous extract of O. mykiss showed potent peroxynitrite radical scavenging activity ($IC_{50}=0.12{pm}0.001;mg/mL$) and reducing power (absorbance=$0.47{pm}0.001$) at the concentration of 1 mg/mL. The in vitro cholinesterase inhibitory activity of O. mykiss aqueous extract was examined using spectrophotometric analyses of acetyl- and butyrylcholinesterase. The aqueous extract of O. mykiss showed acetylcholinesterase inhibitory activity ($IC_{50}=1.61{pm}0.13;mg/mL$), but did not exhibit inhibitory activity against butyrylcholinesterase. These results suggest that O. mykiss possesses antioxidant and acetylcholinesterase inhibitory activities and provide scientific evidence for the health benefits of O. mykiss aqueous extract.