The current study was undertaken to investigate the protective role of melatonin (MEL) and acetyl-L-carnitine (ALC) against dexamethasone (DM)-induced neurotoxicity. Adult female rats (60) were divided into: (1) control group, (2) DM-treated group, (3) MEL-treated group, (4) ALC-treated group, (5) MEL- and DM-treated, and (6) ALC- and DM-treated group. Serum acetylcholinesterase (AchE) activity, malondialdehyde (MDA), nitric oxide (NO) level, catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities were estimated. Gene expression of the prooxidants (NO synthases NOS-1, NOS-2 and heme oxygenases HO-1, HO-2) and antioxidant enzyme (GST-P1) as well as deoxyribonucleic acid (DNA) fragmentation analysis of brain tissue were investigated. Histological examination of the brain tissue was carried out. DM administration caused significant increase in serum AchE activity, MDA and NO levels accompanied with significant decrease in the antioxidant enzymes activity. Pretreatment with MEL or ALC prior DM has been found to reverse all the former parameters. On the genetic level, DM administration significantly increased the expression level of NOS-1, NOS-2, HO-1, and HO-2 messenger ribonucleic acids (mRNAs) and decreased that GST-P1-mRNA in brain tissue. Also, DM produced DNA fragmentation in brain tissue. Treatment with MEL or ALC prior DM administration tend to normalize the above mentioned parameters. These results were documented by the histological examination of brain tissue. The present study suggests that oxidative stress is involved in the pathogenesis of DM-induced neurotoxicity. The inhibition of oxidative stress via stimulation of the antioxidant enzymes by MEL and ALC pretreatment plays a central protective role in modulation of neurotoxicity induced by DM.