Background: There is not enough information about the prevalence of Acinetobacter infection as well as its risk factors, especially in neonatal intensive care units. The present research aimed at conducting a five-year study on Acinetobacter infection and its main factors in neonatal and pediatric intensive care units in Iran. Methods: This cross-sectional survey was conducted on 89 children with positive culture for hospital-acquired Acinetobacter, admitted to intensive care units of Aliasghar Children's Hospital in Tehran, between 2010 and 2015. Besides, 97 patients with similar baseline characteristics without Acinetobacter positivity were enrolled as the control group. Epidemiological information and clinical data were collected by reviewing the hospital recorded files. Results: In the group with positive Acinetobacter culture, complete and partial improvement was observed in 62.9% and 11.2%, respectively, while 25.8% died due to treatment failure. In this regard, complete and partial improvement in the control group was revealed in 85.6% and 11.3% with an overall death rate of 3.1%, indicating significantly higher failure rate in the case group (P = 0.001). To determine the main factors for in-hospital death, all variables with a significant association with positive culture in univariate analysis (considering P < 0.2) were entered in a backward multivariable logistic regression model. In this regard, venous access (OR = 7.80, 95% CI: 1.06 to 57.19, P = 0.043), carbapenem use (OR = 27.03, 95% CI: 1.93 to 377.780, P = 0.014), and ampicillin use (OR = 0.12, 95% CI: 0.019 to 0.739, P = 0.022) were shown as the main determinants for Acinetobacter-related death. Conclusions: Although the study was not a prognostic study and determination of the main determinants of prognosis in children with Acinetobacter infections was not possible yet it seems that the mortality rate due to Acinetobacter infection in the population was about 25.8% in the global range reported in the literature. The main factors for Acinetobacter infection-related death are central venous catheters related to TPN, carbapenem use, and ampicillin use.