Point Prevalence Surveys (PPSs) provide useful data on the patterns of in-hospital antimicrobial prescription. Aiming to identify targets for quality improvement, we evaluated prescribing patterns of antimicrobials in the pediatric and neonatal wards of two tertiary referral centers in Iran. Materials and Methods: Two PPSs on antimicrobial use in children and neonates hospitalized in the Nemazee teaching hospital in Shiraz (south of Iran) and Besat teaching hospital in Sanandaj (west of Iran) were performed for two consecutive years. We used a validated and standardized method based on the Antibiotic Resistance and Prescribing in European Children project. Results: Out of a total of 266 and 129 admissions in pediatric and neonatal wards, respectively, 61% of pediatric inpatients and 71% of neonates received at least one antimicrobial. The most frequently prescribed antibiotics in pediatric wards were ceftriaxone (29.2%) and vancomycin (15%), and in neonatal wards, ampicillin (34.7%) and cefotaxime (14.7%). Antimicrobial combination therapies and the parenteral route of administration in pediatric wards were 40% and 91.3%, and in neonatal wards, 63% and 100%, respectively. Empirical antibiotic therapies in pediatric and neonatal wards were 93.6% and 96%, respectively. Conclusion: The high percentage of antimicrobial use, combination therapies, and empirical therapies could be the targets for quality improvement in our hospitals.