The widespread presence of microplastics (MPs) in freshwater systems has raised concerns about their potential ecotoxicity on aquatic organisms. In this study, we evaluated the effects of four MPs with different compositions, namely polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC) and polypropylene (PP), on freshwater microalgae Chlamydomonas reinhardtii. PS and PVC MPs caused greater growth inhibition and stronger oxidative stress response than PP and PE MPs. Proteomics analysis was employed to explore the mechanical understanding of the composition-dependent toxicity of MPs on microalgae. Proteins involved in photosynthesis processes were identified as contributors to the diverse responses of microalgae to differently composed MPs. Photosynthesis activity of algae, including pigment content and photoprotective response, was determined to reflect the distinct effect of the four MPs. The indicated down-regulated expression of photosynthetic proteins by proteomics analysis was further confirmed using western blot, with PVC and PS showing greater impacts on their expression reduction. Our findings not only show the composition-dependent effect of MPs on microalgae but also provide important insights into the molecular mechanism of MPs’ toxicity on natural phytoplankton species.