Concerns have been raised regarding the ecotoxicity of ionic liquids (ILs) owing to their wide usage in numerous fields. Three imidazolium chloride ILs with different numbers of methyl substituents, 1-decyl-imidazolium chloride ([C10IM]Cl), 1-decyl-3-methylimidazolium chloride ([C10MIM]Cl), and 1-decyl-2,3-dimethylimidazolium chloride ([C10DMIM]Cl), were examined to assess their effects on growth, photosynthesis pigments content, chlorophyll fluorescence, photosynthetic and respiration rate, and cellular ultrastructure of Scenedesmus obliquus. The results showed that algal growth was significantly inhibited by ILs treatments. The observed IC50,48h doses were 0.10 mg/L [C10IM]Cl, 0.01 mg/L [C10MIM]Cl, and 0.02 mg/L [C10DMIM]Cl. The chlorophyll a, chlorophyll b, and total chlorophyll content declined, and the chlorophyll fluorescence parameters, minimal fluorescence yield (F0), maximal fluorescence yield (Fm), maximum quantum yield of PSII photochemistry (Fv/Fm), effective quantum yield of PSII [Y(II)], non-photochemical quenching (NPQ) and non-photosynthetic losses yield [Y(NO)] were notably affected by ILs in a dose-dependent manner. ILs affected the primary photosynthetic reaction, impaired heat dissipation capability, and diminished photosynthetic efficiency, indicating negative effects on photosystem II. The photosynthetic and respiration rates of algal cells were also reduced due to the ILs treatments. The adverse effects of ILs on plasmolysis and chloroplast deformation were examined using ultrastructural analyses; chloroplast swelling and lamellar structure almost disappeared after the [C10MIM]Cl treatment, and an increased number of starch grains and vacuoles was observed after all ILs treatments. The results indicated that one-methyl-substituted ILs were more toxic than non-methyl-substituted ILs, which were also more toxic than di-methyl-substituted ILs. The toxicity of the examined ILs showed the following order: [C10IM]Cl < [C10DMIM]Cl ≤ [C10MIM]Cl.