The ecological success of diatoms requires a remarkable ability to survive many types of stress, including variations in temperature, light, salinity, and nutrient availability. On exposure to these stresses, diatoms exhibit common responses, including growth arrest, impairment of photosynthesis, production of reactive oxygen species, and accumulation of triacylglycerol (TAG). We studied the production of cyclopentane oxylipins derived from fatty acids in the diatom Phaeodactylum tricornutum in response to oxidative stress. P. tricornutum lacks the enzymatic pathway for producing cyclopentane-oxylipins, such as jasmonate, prostaglandins, or thromboxanes. In cells subjected to increasing doses of hydrogen peroxide (H2O2), we detected nonenzymatic production of isoprostanoids, including six phytoprostanes, three F2t-isoprostanes, two F3t-isoprostanes, and three F4t-neuroprostanes, by radical peroxidation of α-linolenic, arachidonic, eicosapentaenoic, and docosahexanoic acids, respectively. H2O2 also triggered photosynthesis impairment and TAG accumulation. F1t-phytoprostanes constitute the major class detected (300 pmol per 1 million cells; intracellular concentration, ∼4 µm). Only two glycerolipids, phosphatidylcholine and diacylglycerylhydroxymethyl-trimethyl-alanine, could provide all substrates for these isoprostanoids. Treatment of P. tricornutum with nine synthetic isoprostanoids produced an effect in the micromolar range, marked by the accumulation of TAG and reduced growth, without affecting photosynthesis. Therefore, the emission of H2O2 and free radicals upon exposure to stresses can lead to glycerolipid peroxidation and nonenzymatic synthesis of isoprostanoids, inhibiting growth and contributing to the induction of TAG accumulation via unknown processes. This characterization of nonenzymatic oxylipins in P. tricornutum opens a field of research on the study of processes controlled by isoprostanoid signaling in various physiological and environmental contexts in diatoms.