In the present study, electron paramagnetic resonance coupled with spin-trapping technique was used, with alpha-phenyl-N-tert-butylnitrone (PBN) as a spin-trapping agent, to investigate free radical generation in freshwater fish with acute 2,4-dichlorophenol (2,4-DCP) poisoning. The PBN-radical adducts were detected in fish liver samples following treatments of 2,4-DCP (0.025, 0.05, 0.5, 5, or 25 mg/kg) 24 h after intraperitoneal (i.p.) injection and 2,4-DCP (0.5 mg/kg) at 2, 4, 8, 24, or 72 h after i.p. injection in Carassius auratus. The hyperfine splitting constants for the PBN-radical adducts are aN = 13.7 G, aH = 1.8 G, and g = 2.0058, which is consistent with those of PBN/hydroxyl radical (*OH). The results indicate that the hydroxyl radical is probably produced during acute intoxication of 2,4-DCP. The relative similarity in the kinetics (from 2 to 72 h) of superoxide dismutase activity induction and *OH generation implies that the generation of *OH possibly depends on the superoxide anion (O2*-). Superoxide anion (O2*-) might be the precursor radical undergoing the Haber-Weiss reaction to form *OH. Possible pathways for radical chain reactions in the formation of the hydroxyl radical in vivo after 2,4-DCP administration are proposed. Other parameters with respect to antioxidant defense (e.g., superoxide dismutase and catalase) and oxidative damage (lipid peroxidation level) indicate that the fish were subjected to oxidative stress induced by 2,4-DCP and that the mechanisms of oxidative stress possibly involve the in vivo stimulation of hydroxyl radical formation.