Use of copper sulfate pentahydrate for fungus control on fish embryos is extremely effective and economical for farmers in waters with moderate or high alkalinity/hardness; however, fry toxicity in low alkalinity/hardness waters is a concern. In the present study, the acute toxicity of CuSO4 to Largemouth Bass (LMB) fry in naturally soft water (i.e., 42 mg/L alkalinity, 41 mg/L hardness) was determined. Median lethal concentration (LC50) values at 24 h were 3.97 mg/L CuSO4 for yolk-sac fry and 5.24 mg/L CuSO4 for swim-up fry. Most importantly for farmers, the No Observed Effect Concentrations (NOEC) in the present study was 0.625 mg/L CuSO4 for both stages of fry. In regard to the safe use of CuSO4 for fungus control on LMB embryos, this research was done to demonstrate CuSO4 toxicity to LMB fry in soft water.