Acinetobacter baumannii (A.baumannii) is an opportunistic pathogen that causes serious types of infections in human. It shows a wide range of antibiotic resistance. Objective: to investigate biofilm formation and its relation to biofilm-related genes in multidrug-resistant (MDR) A.baumannii isolated from patients hospitalized in Benha University Intensive Care Unit (ICU). Methodology: This study was conducted on (90) clinical samples obtained from immunocompromised patients with various symptomatic clinical infections, at least 48 hours after admission to Benha University ICU. A. baumannii isolates were identified, their antibiotic susceptibility pattern was detected using VITEK® 2 system. Biofilm-forming capacity of A. baumannii isolates was determined by tube method. Biofilm-related genes (csuE , omp A, bap , bla PER-1, and aba I) were detected using polymerase chain reaction(PCR). Results: Out of 60 A.baumannii isolates, 93.3% were MDR, 76.7% were extensive drug resistant (XDR), and 75% were biofilm formers. A significant relationship was revealed between the multidrug resistance and biofilm formation. Detected ratio of csuE, omp A, bap, bla PER-1,, and aba I genes were 96.7%, 48.3% , 68.3% , 40% and 88.3% respectively. (csu E, bap, aba I, and bla PER-1) genes were founded to have a statistically significant relation to biofilm formation. While (omp A) gene was insignificantly related to biofilm formation. Conclusion: Biofilm formation in A. baumannii is significantly related to multidrug resistance, with high frequency of biofilm -related genes