The prediction of combined effects based on the effects of the individual components of mixtures by using the pharmacological concepts of concentration addition and independent action might be a promising tool for the risk assessment of pollutant mixtures. To analyze and compare the predictive capabilities of the reference concepts for similarly acting chemicals, the overall toxicity of a multiple mixture was determined in a bioluminescence inhibition assay with Vibrio fischeri. The mixture was composed of 16 similarly and specifically acting chemicals, anticipated to have a common mode of action via weak acid respiratory uncoupling of oxidative phosphorylation. Results show that the observed mixture toxicity is rather well predicted by both concepts. Concentration addition shows an excellent predictive power; the median effective concentration (EC50) of the mixture is predicted with an error of about 10%. Independent action, in contrast, underestimates the EC50 of the mixture by a factor of a little more than three. With respect to risk assessment procedures, it may be concluded that concentration addition gives a valid estimation of the overall toxicity for multiple mixtures with similar and specific mechanisms of action of the mixture components in this type of biotest.