Diazinon is an organophosphorous pesticide with a prominent toxicity on many body organs. Multiple mechanisms contribute to diazinon-induced deleterious effects. Inhibition of acetyl-cholinesterase, cholinergic hyperstimulation, and formation of reactive oxygen species may play a role. On the other hand, melatonin is a pineal hormone with a well-known potent antioxidant activity and a remarkable modulatory effect on many behavioral processes. The present study revealed that oral diazinon administration (25 mg/kg) increased anxiety behavior in rats subjected to elevated plus maze and open-field tests possibly via the induction of changes in brain monoamines levels (dopamine, norepinephrine, and serotonin). Additionally, brain lipid peroxides measured as malondialdehyde (MDA) and tumor necrosis factor alpha (TNF-α) levels were elevated, while the activity of brain glutathione peroxidase enzyme was reduced by diazinon. Co-administration of oral melatonin (10 mg/kg) significantly attenuated the anxiogenic activity of diazinon, rebalanced brain monoamines levels, decreased brain MDA and TNF-α levels, and increased the activity of brain glutathione peroxidase enzyme.