Acinetobacter baumannii is a common cause of nosocomial infections. This bacterium is able to survive in hostile environments (desiccation, antimicrobial therapies, nutrient unavailability) beside colonization biotic and abiotic surfaces and form biofilm in hospitals and long-term care institutions. Biofilm is a three-dimensional structure of a multicellular complex in which the cells are embedded in an Extracellular Polymeric Substance (EPS) that was produced by the organism itself. Biofilm matrix contains proteins, ions, nucleic acids, and polysaccharide polymers. The main factor that leads to biofilm formation in A. baumannii is the creation of the exopolysaccharide poly-1,6-N-acetylglucosamine (PNAG), which is a virulence factor required for adhesion and aggregation by many Gram-negative bacteria. Many studies indicated that PNAG is an important factor to keep the integrity of A. baumannii biofilms in a more dynamic and stressful environment. A cluster of four genes (pgaA, pgaB, pgaC, and pgaD) are responsible for PNAG, which is encoded by pgaABCD locus. Therefore, the aim of this work was to detect proteins encoded by this locus using SDS-PAGE gel in A. baumannii isolates. Using PCR analysis to detect 16S rRNA concluded that all 24 isolates showed a positive amplification with 242bp. Acinetobacter baumannii clinical isolates showed high resistance percentage to ampicillin-sulbactam (AMS) and ceftazidime (CAZ) with 100% and 91% respectively. Furthermore, the isolates showed 83.3% for cefepime (FEP) and 70.8% for amikacin (AK), while the isolates showed a variable resistance percentage toward other antibiotics. By using Congo red method indicated that 66.7% were positive to produce biofilm and 33.3 were non-forming biofilm. Protein sequences alignment showed 99%, 99%, 100% and 99% identity for PgaA, PgaB, PgaC and PgaD respectively with protein GenBank database.