It is vital to address potential risks to aquatic ecosystems exposed to runoff and leachates from biochar-amended soils, before large scale applications can be considered. So far, there are no established approaches for such an assessment. This study used a battery of bioassays and representative aquatic organisms for assessing the acute toxicity of water-extractable fractions of biochar-amended soil, at reported application rates (80 t ha−1). Biochar-amended aqueous soil extracts contained cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), manganese (Mn), zinc (Zn), nickel (Ni), lead (Pb), arsenic (As) and mercury (Hg) (Σmetals 96.3 µg l−1) as well as the 16 priority PAHs defined by the U.S. Environmental Protection Agency (Σ16PAHs 106 ng l−1) at contents in the range of current EU regulations for surface waters. Nevertheless, acute exposure to soil-biochar (SB) extracts resulted in species-specific effects and dose–response patterns. While the bioluminescent marine bacterium Vibrio fischeri was the most sensitive organism to aqueous SB extracts, there were no effects on the growth of the microalgae Pseudokirchneriella subcapitata. In contrast, up to 20 and 25 % mobility impairment was obtained for the invertebrate Daphnia magna upon exposure to 50 and 100 % SB extract concentrations (respectively). Results suggest that a battery of rapid and cost-effective aquatic bioassays that account for ecological representation can complement analytical characterization of biochar-amended soils and risk assessment approaches for surface and groundwater protection.