The toxicity of single and combined formulated herbicides (Mikado® and Viper®) was assessed on several endpoints in species from two trophic levels: algae growth—Pseudokirchneriella subcapitata and Chlorella vulgaris—immobilization and life-history traits (only for single compound toxicity) of daphnids—Daphnia longispina and Daphnia magna. Viper was the most toxic formulated herbicide. It was hypothesized that the toxicity of both formulated herbicides could have been enhanced by adjuvants, especially for Viper. In most cases, the sublethal endpoints were the most sensitive and affected by both formulations, comparatively to their acute effects. Concentration addition (CA) and independent action (IA) models provided an accurate description of Mikado and Viper joint action on algae growth and immobilization of daphnids, although significant deviations were always detected. A low-dose antagonism and high-dose synergism were identified for P. subcapitata, whereas C. vulgaris response deviated antagonistically from CA and synergistically from IA. For both daphnids, however, synergistic effects were observed for higher mixture concentrations. Under a regulatory standpoint, CA provided the most conservative estimation either because the mixture effects were overestimated or less subestimated than IA. Overall, the great sensitivity differences observed within species did not allow the conclusion that one trophic level was more tolerant than the other. Instead, P. subcapitata was always the most sensitive species to both herbicide formulations, followed by D. longispina, while D. magna and C. vulgaris were the most tolerant species. On a whole, further studies are needed toward a comprehensive understanding of herbicides mode of action, their effects at lower biological-level endpoints, and under different mixture designs. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2012.