Biodiesel is considered an important renewable energy source but still there is some controversy about its environmental toxicity, especially to aquatic life. In our study, the toxicity of water soluble fraction of biodiesel was evaluated in relatively low concentrations using a battery of bioassays: Vibrio fischeri bioluminescence inhibition, Sinapis alba root growth inhibition, Daphnia magna immobilization, boar semen live/dead ratio and DNA fragmentation and Unio pictorum micronucleus test. While the S. alba test indicated nutritive (stimulating) effect of the sample, the biodiesel exerted toxic effect in the aquatic tests. D. magna was the most sensitive with EC50 value of 0.0226%. For genotoxicity assessment, the mussel micronucleus test (MNT) was applied, detecting considerable genotoxic potential of the biodiesel sample: it elucidated micronuclei formation already at low concentration of 3.3%. Although this test has never been employed in biodiesel eco/genotoxicity assessments, it seems a promising tool, based on its appropriate sensitivity, and representativity.