Objectives: Perinatal asphyxia (PA) is very significant in perinatal medicine due to the involvement of the central nervous system. This study was conducted to investigate the biochemical, clinical, and paraclinical changes associated with phenobarbital administration in neonates with PA. Methods: In this prospective, case-control study, 30 neonates with PA in two groups of 15 each (case and control) were investigated. The case group received 20 mg/kg intravenous phenobarbital within six hours of birth, and the control group did not receive phenobarbital. Serum concentrations of nitric oxide (NO) were measured at enrollment and one week after birth in the two groups. Clinical, electroencephalography, and magnetic resonance imaging findings of the two groups were compared. Results: At enrollment, the two groups did not differ in clinical severity, seizure incidence, or NO concentration. After one week, NO concentration was significantly lower in the case group (p < 0.050), but there was no significant difference in other variables between the two groups. Conclusions: Early administration of phenobarbital in term neonates with PA could protect them against encephalopathy.