Anesthetic efficacy, plasma cortisol concentration, and two parameters of neutrophil function (oxidative burst and degranulation of primary granules) were compared among three anesthetics in the fathead minnow: tricaine methanesulfonate (MS 222), metomidate hydrochloride (MTMD), and eugenol (EUG). The optimum anesthetic concentration was determined as: MS 222 75 mg L− 1, EUG 30 mg L− 1 and MTMD 4 mg L− 1. Handling and crowding stress was induced in fish with (SA) and without (S) anesthetic. Plasma cortisol concentration was measured at 0, 30, 90, and 240 min after stress and found to increase at 30 min post-stress in S and SA MS 222 groups, but not in SA MTMD and SA EUG groups. To test the effects of different anesthetics on neutrophil function, fish were divided into a baseline control group, a group exposed to handling and crowding stress (S) and a stressed anesthetized group (SA). Fish were assayed for neutrophil function before and after stress (24 h, 72 h and 7 days). The degranulation of neutrophil primary granules was measured as exocytosis of myeloperoxidase (MPO) using 3, 3′, 5, 5′-tetramethylbenzidine as a substrate. Degranulation of primary granules was decreased to 60–75% of non-stressed control in stressed and fish treated with MS 222, and was not affected when MTMD and EUG were used. The degranulation of primary granules proved to be a useful assay for measuring the effects of stress on neutrophil function in fish. Eugenol and metomidate prevented stress-induced decrease of neutrophil function while MS 222 did not.