Acinetobacter baumannii is one of the most important nosocomial, multidrug resistant pathogens. The most important virulence factor is its ability to form biofilms. Microtiter-plate method was used to detect the ability of twenty A. baumannii clinical strains to form biofilm in polystyrene wells. Biofilm formation was assessed by staining with a crystal violet solution 0.1% then elution by an ethanol-acetone mixture (80:20) and O.D measurement. In general, among the twenty isolates, 2 isolates were strong biofilm formers, 13 were medium and 5 were weak. It is worthy to mention that the strong biofilm forming strains were those isolated from tracheal aspirates and wound swabs. When examining environmental factors' effects on biofilm formation, results revealed that biofilm formation was enhanced in the presence of lactose as a carbon additive. Production of biofilm was maximum at pH 6 and 7.4 as indicated from O.D values. Biofilm formation was better at all temperatures with a slight increase at 40oC and was maximum after 72h. The effect of addition of serum on biofilm formation varies among the isolated samples giving rise to high O.D values in sample 2 and 4. Non-biofilm producing mutant generated by 70% CV treatment of strain no. 1 was tested and found to be as antibiotic resistant as the wild type strains indicating the contribution of other factors in addition to biofilm to resist antibiotics.