ABSTRACT: This study was conducted in order to assess the acquisition rate of Acinetobacter baumannii by newborn screening, on admission and during the discharge process of neonatal intensive care unit. (NICU). Furthermore, we investigated risk factors for potential colonization and molecular epidemiology of isolated resistant bacteria. This prospective study was conducted in the neonatal unit of Hassan II University Hospital of Fez from February 2013 to July 2015. During this period, all consecutive admitted neonates were screened for A. baumannii intestinal carriage, on admission and during the discharge process. Bacteriological and molecular tests were evaluated according to the international standards. This study examines the screening on admission of 455 newborns, 59% of whom were male. The average gestational age and birth weight were 35.2 weeks and 2612.1 g respectively. In total, 277 patients were included in the acquisition study on admission. The prevalence of multi-drug resistant (MDR) A. baumannii strain carriage was 6.5%, while the acquisition rate during the hospital recovery was 13.7%. In this study, 68 MDR A. baumannii isolates were collected. The resistance rates to different antibiotic classes including, Ceftazidime, Gentamycin and Ciprofloxacin varied between 92 and 100%. Moreover, 13% of MDR A. baumannii isolates were carbapenemase producers and 88% harbored blaOXA-23 gene. On admission, three risk factors were significantly associated with A. baumannii colonization: age (OR, 2.803; IC95%, 1.191-6.596; P = 0.01), gender (OR, 0.382; IC95%, 0.158-0.921; P = 0.03) and the delivery birth at the Maternity of University Hospital (MUH), (OR, 0.196; IC95%, 0.071-0.540; P = 0.002). However during hospitalization, the only risk factor associated with acquisition of A. baumannii was the respiratory distress (OR, 2.270; IC95%, 1.055-4.881; P = 0.03). A high intestinal carriage rate of A. baumannii and multiple antibiotic resistance were found in our NICU. Thus, the spread of MDR A. baumannii should be monitored by an active surveillance strategy.