Tris(1,3-dichloro-2-propyl)phosphate (TDCPP) is one of the most widely used organophosphate ester flame retardants. The presence of TDCPP in surface waters and aquatic organisms have been reported worldwide, yet the ecological risk of TDCPP on microalgae is rarely studied. We investigated the biotransformation of TDCPP and its toxicity on the microalga Scenedesmus obliquus using an untargeted metabolomics approach. Exposure to TDCPP resulted in a dose-response decrease of micoalgal biomass. In the presence of microalgae, TDCPP concentration in the media decreased by 25.3–40.6% after 5 days. TDCPP metabolites were identified in the media including hydrolysis and hydroxyl-substituted dechlorination products. A dose-response separation of metabolic profiles of microalgae was observed, with effect seen at the lowest concentration of 10 µg/L tested, which is slightly higher than environmentally relevant concentrations. Differentiated metabolites identified include 52 lipids and 6 polar metabolites. Analysis of altered lipid pathways suggests that microalgal cells reinforce thylakoid membranes (function to protect photosynthesis) by compromising the integrity of plasma membrane (function to protect cellular substances) and extraplastidial cellular membranes. Changes in the polar metabolites might indicate osmotic stress and improved NO signaling after TDCPP exposure. Consistent with perturbation of membrane lipids, further experiment confirmed that exposure to 10 mg/L TDCPP resulted in significant (p < 0.01) plasma membrane damage. This study indicates biotransformation and the membrane damage toxicity mechanism of TDCPP on S. obliquus, demonstrating the usefulness of metabolomics for the toxicity mechanism elucidation of emerging pollutants.