Effective anesthetics are a critical component of safe and humane fish handling procedures. We tested three concentrations each of four anesthetics—Finquel (tricaine methanesulfonate, herein referred to as MS-222), BENZOAK (20% benzocaine), AQUI-S 20E (10% eugenol), and Aquacalm (metomidate hydrochloride)—for efficacy and safety in metamorphosed, outmigrating juvenile Pacific Lampreys Entosphenus tridentatus. The anesthetics MS-222 (100 mg/L) and BENZOAK (60 mg/L) were the most effective for anesthetizing juvenile Pacific Lampreys to a handleable state with minimal irritation to the fish. Fish anesthetized with BENZOAK also had lower rates of fungal infection than those exposed to MS-222, AQUI-S 20E, or no anesthetic. Exposure to AQUI-S 20E irritated juvenile Pacific Lampreys, causing them to leap or climb out of the anesthetic solution, and Aquacalm anesthetized fish to a handleable state too slowly and incompletely for effective use with routine handling procedures. Our results indicate that MS-222 and BENZOAK are effective anesthetics for juvenile Pacific Lampreys, but field studies are needed to determine whether exposure to MS-222 increases risk of fungal infection in juvenile Pacific Lampreys released to the wild.