As a ubiquitous contaminant in aquatic environments, diethyl phthalate (DEP) is a major threat to ecosystems because of its increasing utilization. However, the ecological responses to and toxicity mechanisms of DEP in aquatic organisms remain poorly understood. To address this environmental concern, we selected Chlorella vulgaris (C. vulgaris) as a model organism and investigated the toxicological effects of environmentally relevant DEP concentrations at the individual, physiological, biochemical, and molecular levels. Results showed that the incorporation of DEP significantly inhibited the growth of C. vulgaris, with inhibition rates ranging from 10.3 % to 83.47 %, and disrupted intracellular chloroplast structure at the individual level, while the decrease in photosynthetic pigments, with inhibition rates ranging from 8.95 % to 73.27 %, and the imbalance of redox homeostasis implied an adverse effect of DEP at the physio-biochemical level. Furthermore, DEP significantly reduced the metabolic activity of algal cells and negatively altered the cell membrane integrity and mitochondrial membrane potential. In addition, the apoptosis rate of algal cells presented a significant dose–effect relationship, which was mainly attributed to the fact that DEP pollutants regulated Ca2+ homeostasis and further increased the expression of Caspase-8, Caspase-9, and Caspase-3, which are associated with internal and external pathways. The gene transcriptional expression profile further revealed that DEP-mediated toxicity in C. vulgaris was mainly related to the destruction of the photosynthetic system, terpenoid backbone biosynthesis, and DNA replication. Overall, this study offers constructive understandings for a comprehensive assessment of the toxicity risks posed by DEP to C. vulgaris. Display omitted •Exploring the potential toxic mechanisms of DEP on microalgae from multiple angles•DEP initiates the antioxidant system of microalgae by activating SOD and POD viability.•The apoptosis pathway (internal and external pathway) is discussed.•DEP interferes with photosynthesis-antenna proteins and DNA replication in microalgae.