Hatchery personnel depend on therapeutant treatments to control diseases. Currently, hatchery managers in the United States are limited to one approved therapeutant (formalin) and three compounds of Low Regulatory Priority (sodium chloride, hydrogen peroxide, and acetic acid) to control external diseases of cultured fish. Hydrogen peroxide has been used to effectively control external columnaris and bacterial gill disease in rainbow trout, however, definitive safe treatment concentrations for hydrogen peroxide are lacking for a variety of species. We report the acute toxicity of hydrogen peroxide treatments to 11 species of fry and 13 species of fingerling freshwater fish. Most mortality occurred within the first 30 h after the first exposure to hydrogen peroxide with little change in the overall shape of survival curves over time. Our data predict that in an actual therapeutic application of hydrogen peroxide, most treatment-related mortalities would be observed shortly after the initial exposure. Coolwater species were more sensitive than coldwater species but were generally similar to warmwater species tested. Based on our mortality data, coldwater species and largemouth bass may be treated for 60 min at concentrations of ≤ 150 (μl/1 without harmful effects; all muskellunge, walleye, bluegill, channel catfish, yellow perch, pallid sturgeon fingerlings, fathead minnow fingerlings, white sucker fingerlings, and northern pike fry may be treated for 60 min at ≤ 100 μl/l; and northern pike fingerlings and white sucker, yellow perch and fathead minnow fry may be treated for 60 min at ≤ 50μl/l.