Ionic liquids (ILs) are widely used, but their potential threat to the environment has recently gained more attention. The enantioselective oxidative stress caused by chiral ionic liquids (CILs), such as 1-alkyl-3-methyl imidazolium tartrate (RMIM T), on Scenedesmus obliquus was demonstrated in this study. Stronger green fluorescence was observed in response to l-(+)-RMIM T treatment than to d-(+)-RMIM T treatment, which suggested that more reactive oxygen species (ROS) were stimulated by l-(+)-RMIM T. Significantly higher ROS levels were recorded during the RMIM T treatments than in the control. There were 1.13-, 1.25-, 1.43-, 1.68-, and 1.96-fold increases over levels in the control in the 3, 5, 10, 15, and 25mg/L d-(−)-HMIM T treatments, respectively, and 1.26-, 1.37-, 1.58-, 1.86- and 2.08-fold increases over levels in the control in the 3, 5, 10, 15, and 25mg/L l-(+)-HMIM T treatments, respectively. The total soluble protein content decreased as the RMIM T concentration increased. The SOD and CAT activities were stimulated at lower concentrations, but were inhibited at higher concentrations. Regression analysis implied that ROS is the major factor responsible for the oxidative damage caused by RMIM T. The ultrastructural morphology analysis showed that plasmolysis and damage to the chloroplasts, starch granule decreases, and lipid granule increased, and pyrenoid and nucleoid damage had occurred. These results showed that enantioselective oxidative stress and oxidative damage were caused by d-(+)-RMIM T and l-(+)-RMIM T, and that l-(+)-RMIM T caused more damage than d-(+)-RMIM T.