Dimethyl sulfoxide (DMSO) is a universally used aprotic solvent with the ability to permeate biological membranes and thus is commonly used to achieve appropriate biological availability of hydrophobic toxicants. While DMSO as a carrier medium has a reportedly low toxicity and is routinely employed in ecotoxicology, very little is known about its effect on dynamic behavioral parameters. This study presents a comparative analysis of the lethal and behavioral effects of exposures to DMSO concentrations of 0.1–10% on several test species such as: neonates of the freshwater crustacean Daphnia magna, nauplii of the marine crustacean Artemia franciscana, the marine crustacean Allorchestes compressa, embryos and larvae of the freshwater fish Danio rerio. The results demonstrated that DMSO did not cause statistically significant mortality even at concentrations close to 1% but induced clear and significant behavioral abnormalities in response to sublethal concentrations on all test species. These included hypoactivity syndrome in A. franciscana, A. compressa, D. magna and zebrafish larvae while a slight time-dependent hyperactivity response was observed in zebrafish embryos. For the majority of test species, behavioral changes such as moving distance, acceleration and burst movement were often observed during the first hours of exposure. These results indicate that caution should be exercised when using DMSO as a carrier solvent in experiments assessing behavioral endpoints.