Background: Acinetobacter baumannii, a nosocomial pathogen, causes severe ICU infections. It's a non-fermentative, gram-negative bacterium. This study aimed to evaluate PCR's efficiency for rapid A. baumannii diagnosis, detect biofilm formation, and assess antimicrobial susceptibility in ICU-isolated A. baumannii. Methods: This cross-sectional study involved 40 isolates of Acinetobacter baumannii bacteria from patients who developed clinical evidence of nosocomial infection. Different types of specimens were collected as follows: Lower respiratory tract specimens (25), Blood samples (10), Urine samples (3) and pus samples from infected surgical and traumatic wounds (2). Diagnosis is done by conventional methods, BD phoenix M50 and PCR for detection of specific sequence of Acinetobacter baumannii (gyrB gene sequences). Biofilm formation was assessed as a virulance factor using 96-well microtiter plates and tube method. Results: PCR and BD Phoenix™ M50 both showed high agreement with culture for detecting Acinetobacter, scoring perfect (1) and excellent (0.979) respectively. PCR demonstrated 100% accuracy, NPV, PPV, specificity, and sensitivity, while Phoenix showed 98.4 NPV, 100% accuracy, specificity, PPV, and 97.5% sensitivity. At the genus level, their agreement was 0.979, and at the species level, it was 0.958. The gyr B gene appeared in all 40 A. baumannii isolates. The most effective antibiotic was trimethoprim-sulfamethazole (37.5%) for acinetobacter infections. Biofilm formation occurred in 52.5% of cases via plate and tube methods. Conclusion: PCR and BD Phoenix M50 are rapid, specific, reliable and easy to interpret methods for accurate detection of Acinetobacter baumannii.