The increasing interest in the development of novel green solvents has led to the synthesis of benign alternative products with minimized environmental impacts. However, most of published studies on green solvents focus primarily on their physicochemical properties, with limited emphasis on absence of ecotoxicological assessment. In this study, we evaluated the acute ecotoxicity of four levulinates (levulinic acid, methyl levulinate, ethyl levulinate and butyl levulinate) on freshwater algae (Chlamydomonas reinhardtii), bacteria (Vibrio fischeri), daphnids (Daphnia magna) and earthworms (Eisenia foetida) using various dose–response tests. As a general trend, the toxicity of levulinate esters in aquatic exposure (assessed as the EC₅₀) increased as a function of increasing alkyl chain length; accordingly, the most toxic compound for the aquatic organisms was butyl levulinate, followed by ethyl levulinate and methyl levulinate. The most toxic compound for E. foetida (terrestrial exposure) was methyl levulinate, followed by ethyl levulinate, butyl levulinate and levulinic acid; in this case, we observed an inverse relationship between toxicity and alkyl chain length. Based on both the lowest EC₅₀ found in the aquatic media and the ratio between predicted environmental concentration and the predicted no-effect concentration, we have estimated the maximum allowable values in the environment for these chemicals to be 1.093 mg L⁻¹ for levulinic acid, 2.761 mg L⁻¹ for methyl levulinate, 0.982 mg L⁻¹ for ethyl levulinate and 0.151 mg L⁻¹ for butyl levulinate.