Previous research evaluated the toxicity of the lampricide 4-nitro-3-(trifluoromethyl)phenol (TFM) and the combination of TFM with 1 % niclosamide (TFM:1%Nic) to multiple non-target species in the Laurentian Great Lakes. However, few toxicity studies have been conducted for species of concern in Lake Champlain (NY and VT). We conducted 12-hour flow-through toxicity tests with 4 species of native mussels, 6 species of fish, and 1 amphibian species. All tests included exposure of invasive larval Petromyzon marinus (sea lamprey) and were conducted with concentrations that bracketed the predicted minimum lethal concentration required to control larval sea lamprey. Mussel species’ NOEC, LOEC, LC₂₅, and LC₅₀ values ranged from 1.33 to 2.12, 1.71–2.66, 1.75–3.05, and 2.03–4.84 times field determined LC_99.9s for sea lamprey (×SL_LC99.9) in TFM-only toxicity tests, and from 1.36 to 1.70, 1.68–2.03, 1.86–2.10, and 2.35–2.68 × SL_LC99.9 for TFM:1%Nic toxicity tests, respectively. Fish species NOEC, LOEC, LC₂₅, and LC₅₀ values ranged from 0.60 to 1.89, 0.73–2.13, 0.72–2.11, and 0.76–2.18 × SL_LC99.9 in TFM-only toxicity tests, and from 0.64 to 2.48, 0.85–3.10, 0.74–3.05, and 0.78–3.16 × SL_LC99.9 for TFM:1%Nic toxicity tests, respectively. Amphibian species NOEC, LOEC, LC₂₅, and LC₅₀ values ranged from 0.74 to 0.75, 0.85–0.95, 0.83–0.87, and 0.85–0.91 × SL_LC99.9 in TFM-only toxicity tests, and from 0.63 to 0.65, 0.80–0.88, 0.77–0.82, and 0.78–0.87 × SL_LC99.9 for TFM:1%Nic toxicity tests, respectively. Generally, mussel species were tolerant, fish sensitivities were variable, and the amphibian species was sensitive to TFM and TFM:1%Nic.