Abstract: As an antibacterial agent, trimethoprim (TMP) is frequently detected in water environment, but the existing studies pay more attention to its acute toxicity. In order to accurately assess the risk to aquatic ecosystem, Phaeodactylum tricornutum and Skeletonema costatum were taken as the research objects in this study to explore the toxic effects of environmental concentration TMP on them. The results showed that the activities of superoxide dismutase (SOD) of two species of marine microalgae decreased first and then increased with the extension of exposure time under TMP exposure. TMP also caused changes in glutathione oxidation system in algae cells, including glutathione (GSH) consumption and glutathione peroxidase (GPx) activity. High concentration of TMP caused serious lipid peroxidation in P. tricornutum. The integrated biomarker response(IBR_v2) index is positively correlated with the concentration of TMP, indicating that it can be used to evaluate the ecotoxicological effect of TMP on P. tricornutum. The results are of great significance for evaluating its long-term impact on marine ecosystems and formulating relevant protection measures.