Root exudates of two varieties (Burley and K326) of tobacco seedlings cultured in a hydroponic medium were collected, and identified using gas chromatography-mass spectrometry (GC-MS). Further, the effects of different concentrations of dibutyl phthalate (DBP) and diisooctyl phthalate (DIOP) on root antioxidant capacity were assessed using pot culture experiments. The results revealed three main compounds in Burley root exudates. The relative content of autotoxin phthalate esters (PAEs) at the two-, four-, and six-leaf stages were 7.6%, 0.3%, and not detected, respectively. However, there were nine main compounds in K326 root exudates. The relative PAE content at the two-, four-, and six-leaf stages were 35.6%, 51.3%, and 2.2%, respectively. PAEs such as DIOP and DBP were identified as the major autotoxins in root exudates of both tobacco seedlings. The rate of superoxide anion radical generation in roots significantly increased at concentrations greater than 0.1 mmol/L DIOP and DBP (P < 0.05). Both superoxide dismutase and catalase activities increased with increasing DIOP concentrations, with a peak at 0.5 mmol/L, and subsequently decreased at higher concentrations. Accumulated malondialdehyde concentrations increased with increasing DIOP and DBP concentrations, and the magnitude of malondialdehyde content was DIOP > DBP, which indicated the order of their toxic effect. Finally, our findings also revealed that when PAEs reached 0.5 mmol/L, they decreased the antioxidant capacity of the root system, initiated oxidative damage of the root cell membrane system, and caused a further decrease in root absorption and mineral metabolism, and thereby led to autotoxicity in the tobacco plants.