The present study was designed to understand the effects of piperonyl butoxide (PBO), modulator of cytochrome P450 (CYP 450), on the neurotoxicity of organophosphate pesticide fenthion in the brain of Oreochromis niloticus used as a model organism. Fish were exposed to one-fourth of the LC50 value of fenthion (0.567 mg/L) and 0.5 mg/L PBO concentration for 24 h, 96 h, and 15 days. Glutathione (GSH)-related antioxidant system, lipid peroxidation, stress proteins, and acetylcholinesterase (AchE) activity were investigated. Our results showed that PBO induced the neurotoxic effect of fenthion with increasing oxidative stress in long-term exposure. GSH-related antioxidant system might take a role in protecting the brain from these oxidative effects. PBO possibly inhibited the biotransformation of fenthion by inhibiting CYP 450; thereby preventing the brain from AChE inhibition in short-term exposure. Changes in parameters indicated that PBO caused biphasic response by affecting CYP 450 in the brain of O. niloticus.