The rising prevalence of Acinetobacter infections in the intensive care units (ICUs) causes a great concern due their extraordinary ability to develop resistance to multiple classes of antimicrobial agents. This is a cross-sectional and facility based study that conducted in King Khalid hospital in Najran, Saudi Arabia. The aim of this study is to estimate the prevalence of Acinetobacter infections in the ICU patients, and to identify antibiotic susceptibility of the Acinetobacter isolates. 180 ICU patients were studied during the period from Aug. 2016 to April 2017. Acinetobacter was isolated in 21(11.7%) of patients. It was isolated from tracheal secretions in 43% of the isolates, followed by sputum (23%), wounds (14%), Central Venous Line (CVL) (9%), urine and blood (6%) for each respectively. Acinetobacter infection was increased significantly in patients with old ages, patients with chronic diseases and those undergoing invasive procedures (P=0.001). Acinetobacter sensitivity tests were performed, they showed that the isolates were resistant to many antibiotics in terms of Ampicillin, Cephalothin, Cefuroxime, Gentamycin, Amikacin, Tobramycin, Ofloxacine, Rifampicin and Colistin was respectively 87%, 86%, 79%, 76%, 52%, 43%, 33%, 32% and 8%. It was concluded that Acinetobacter is an important nosocomial infection pathogen in the ICU patients. Few treatment options are currently available, and for this reason, prevention and infection control measures are essential, including antibiotic control strategies in the ICUs.