Dimethyl sulfoxide (DMSO) is widely used as a vehicle solvent in ecotoxicity bioassays.Despite its frequent use, it could be toxic for organisms at some concentrations. Hence, this study aims to investigate the effects of DMSO on the model organisms: microalgae Raphidocelis subcapitata, microcrustacean Daphnia magna, and rotifer Brachionus calyciflorus. DMSO was applied to the organisms in concentrations ranging from 0.031% to 4%. For R. subcapitata the lowest observed effect concentration (LOEC) after 72 h of exposure was 0.125% DMSO. The 50% effective concentration (EC50) was 2.138 ± 0.372%. In D. magna, significant differences in the mobility after 24 h or 48 h of exposure were observed for 1% DMSO being 1.712 ± 0.207% and 1.167 ± 0.220% DMSO the EC50 observed for 24 h and 48 h exposure, respectively. For B. calyciflorus, it was not possible to validate the tests performed, as there were insufficient animals alive in the control conditions at the end of the exposure period. Therefore, we recommended avoiding DMSO as a vehicle in assays using B. calyciflorus, and to use final DMSO concentrations in an experimental solution not exceeding 0.125% for R. subcapitata and 0.5% for D. magna. Finally, using a maximum of 0.01% DMSO is safe for the organisms tested. The effects of the vehicle solvent dimethyl sulfoxide (DMSO) were investigated in three aquatic model organisms, the microalgae Raphidocelis subcapitata, the microcrustacean Daphnia magna and the rotifer Brachionus calyciflorus. As a result, negative effects on growth rate for the algae population, immobilisation of daphnids and inhibition of rotifer reproduction was observed obtaining EC50 close to 1% of DMSO for all these cases.