Background: Hospital-acquired infection is one of the main concerns in Neonatal Intensive Care Units (NICUs), leading to increased mortality, hospital stay, and costs. Objectives: This study aimed to investigate the risk factors of hospital-acquired infection in NICUs. Methods: A descriptive, cross-sectional, prospective study was conducted in the NICU of Ali Asghar Children Hospital for one year. All admitted newborns were sampled on a simple basis. The criteria for the diagnosis of hospital-acquired infection were based on the definitions of the CDC and the NNIS system. Risk factors such as days of fully catheters usage, nurse-to-patient ratio, history of surgery, prematurity, and mechanical ventilation were considered as variables. The data collection tools consisted of a patient information questionnaire, the monthly report of the hospital infection control committee based on the NNIS system, a daily schedule of all risk factors for each infant, and the monthly nurse-to-patient ratio in the NICU. The STATA software was used for data analysis. Results: In our study, 654 newborns were enrolled. The rate of hospital-acquired infections was 13.5%. Moreover, 80.7% of the cases exhibited sepsis (72.7% diagnosed based on clinical findings and 8% based on positive blood culture). Statistical analysis showed 9% pneumonia cases, 8% surgical site infection cases, and 2.3% urinary tract infection cases. The average time to the occurrence of hospital-acquired infection was 13.5 days after admission. All risk factors were significantly higher in the infected group than in the control group (P = 0.0001). Furthermore, surgical interventions were significantly more in the infected group than in the non-infected group (34.1% vs. 6.7%, respectively, P = 0.0001). The prevalence rates in different weight ranges (less than 1000 g, 1001 to 1500 g, 1501 to 2500, and above 2501 g) were 2.6%, 6.9%, 21.4%, and 69.1%, respectively, in the infected group, which were significantly different from those of the non-infected group (P = 0.0001). The most common etiologic microorganism was Acinetobacter baumannii. Conclusions: Factors such as surgery, the presence of a central venous catheter, and the increased length of hospital stay significantly increased the hospital-acquired infections. Reducing invasive procedures, maintenance of full catheters, and providing optimal nursing care can help control hospital-acquired infections.