Background: The association between the use of antibiotics and bacterial resistance has obviously been established and it seems to be a significant problem for public health. It is clear that irrational use and high rates of antibiotic prescription are associated with increased bacterial resistance. As antibiotics are the class of drugs commonly prescribed in neonatal and Neonatal Intensive Care Unit (NICU) wards, neonates are at high risk of opportunistic or nosocomial infections due to prolonged hospitalization and immunosuppressed condition. Objectives: It is essential for antibiotic prescription patterns to be evaluated periodically for rational use. Therefore, the present study was carried out to identify the prescribing patterns of antibiotics in neonatal and NICU wards of 17 different Iranian hospitals. Methods: The study was done during 1 calendar week between January and February 2014. All in-patients admitted to the NICU and neonatal units of 17 Hospitals in 15 Iranian cities were included. Relevant data of all neonates receiving an antibiotic at 8 am on the day of the study was collected by trained members of the study team; age of the patients, type and number of administered antibiotics, route of administration, underlying disease, and indication for use were documented.