The acute toxicity of individual and mixtures of three herbicides (2,4‐D, Garlon‐3A, and Roundup) and a chemical additive (Syndets surfactant) was evaluated using three species of freshwater organisms (channel catfish, bluegill sunfish, and crawfish). Among the three herbicides, Roundup was more toxic to catfish and bluegill than Garlon‐3A and 2,4‐D. The order of toxicity in fish (Roundup > Garlon‐3A > 2,4‐D) was reversed in bioassays with crawfish, indicating a significant difference in interspecies (vertebrate vs invertebrate) biological variability. Adjusted 96 h LC50's were 14.5, 13.0, and 21,632.8 mg/L for Roundup, 109.5, 91.0, and 6,397.5 mg/L for Garlon‐3A, and 181.2, 266.3, and 750.1 mg/L for 2,4‐D, for catfish, bluegill, and crawfish, respectively. For Syndets surfactant, LC50 values of 1.9 mg/L (bluegill), 2.3 mg/L (catfish), and 15.2 mg/L (crawfish) were recorded, indicating that this chemical additive was much more toxic than the three herbicides. Bioassays with herbicide mixtures resulted in combined toxic effects that were slightly antagonistic and/or additive. Lethal concentrations of herbicide mixtures were found to be lower than recommended field formulations, but it was anticipated that such hazardous amounts would be rarely reached in roadside ditches where the presence of large volumes of water is likely to provide dilutions to levels that may not pose a threat to aquatic life. ©1997 by John Wiley & Sons, Inc. Environ Toxicol Water Qual 12: 237–243, 1997