Elevated nitrate (NO3) and sulfate (SO4) in surface water are of global concern, and studies are needed to generate toxicity data to develop environmental guideline values for NO3 and SO4. The present study was designed to fill existing gaps in toxicity databases by determining the acute and/or chronic toxicity of NO3 (tested as NaNO3) to a unionid mussel (Lampsilis siliquoidea), a midge (Chironomus dilutus), a fish (rainbow trout, Oncorhynchus mykiss), and 2 amphibians (Hyla versicolor and Lithobates sylvaticus), and to determine the acute and/or chronic toxicity of SO4 (tested as Na2SO4) to 2 unionid mussels (L. siliquoidea and Villosa iris), an amphipod (Hyalella azteca), and 2 fish species (fathead minnow, Pimephales promelas and O. mykiss). Among the different test species, acute NO3 median effect concentrations (EC50s) ranged from 189 to >883 mg NO3‐N/L, and chronic NO3 20% effect concentrations (EC20s) based on the most sensitive endpoint ranged from 9.6 to 47 mg NO3‐N/L. The midge was the most sensitive species, and the trout was the least sensitive species in both acute and chronic NO3 exposures. Acute SO4 EC50s for the 2 mussel species (2071 and 2064 mg SO4/L) were similar to the EC50 for the amphipod (2689 mg SO4/L), whereas chronic EC20s for the 2 mussels (438 and 384 mg SO4/L) were >2‐fold lower than the EC20 of the amphipod (1111 mg SO4/L), indicating the high sensitivity of mussels in chronic SO4 exposures. However, the fathead minnow, with an EC20 of 374 mg SO4/L, was the most sensitive species in chronic SO4 exposures whereas the rainbow trout was the least sensitive species (EC20 > 3240 mg SO4/L). The high sensitivity of fathead minnow was consistent with the finding in a previous chronic Na2SO4 study. However, the EC20 values from the present study conducted in test water containing a higher potassium concentration (3 mg K/L) were >2‐fold greater than those in the previous study at a lower potassium concentration (1 mg K/L), which confirmed the influence of potassium on chronic Na2SO4 toxicity to the minnow.