Ionic liquids (ILs) are massively used in multiple fields of industry, and consequently, they have entered the environment and become potential threats to the respective ecosystems. In this paper, the toxicity of two different cationic types of ILs (1-hexyl-3-methyl pyridine chloride ([C6Py]Cl) and 1-hexyl-3-methyl imidazole chloride ([C6MIM]Cl)) to Chlorella pyrenoidosa (C. pyrenoidosa) was investigated. Growth inhibition increased with increasing ILs concentrations. C. pyrenoidosa showed a certain recovery at low ILs concentrations, the growth inhibition decreased from 6.13% to 1.57% of the control from 24 h to 96 h, respectively, in 0.5 mg/L [C6MIM]Cl treatment. However, growth inhibition was negatively related with exposure time at high concentrations, and the strongest toxic effects were observed after 48 h. The IC50 values (half inhibitory concentration) were 8.47, 6.65, 6.91 and 7.11 mg/L of [C6MIM]Cl, respectively, in 24, 48, 72, and 96 h, and were 9.05, 6.83, 7.79 and 8.14 mg/L of [C6Py]Cl, respectively. Chlorophyll content declined with higher concentrations of the ILs. The values of chlorophyll fluorescence parameters: the maximum effective quantum yield of photosystem II (PSII) (Fv/Fm), maximum quantum yield in PSII (Fv/F0), and photosynthetic efficiency in PSII (Y(II)), decreased, whereas the minimal fluorescence (F0) increased following the ILs treatment, indicating damage to the photosystem II. [C6MIM]Cl and [C6Py]Cl caused deformation of algae cells, plasmolysis, and damage of the cell membrane and cell wall, and affected organelle structure. Reactive oxygen species (ROS) concentrations increased with higher ILs concentrations from, and superoxide dismutase and catalase activity first increased and then decreased, indicating that the antioxidant defense system was activated to counteract ROS. ROS was the main stress in C. pyrenoidosa induced by ILs, and compared with [C6Py]Cl, [C6MIM]Cl were more toxic to C. pyrenoidosa.