Acinetobacter baumannii is a Gram-negative nosocomial pathogen. It is responsible for a variety of infections and is among the five most common pathogens in U.S. hospitals associated with ventilator-associated pneumonia. It has been estimated that A. baumannii is responsible for approximately 45,000 hospital infections in the U.S. per year. The two main characteristics that make this organism a formidable threat in health care settings is its ability to acquire multidrug resistance and to persist on surfaces. A better understanding of the genetic factors responsible for these virulence traits is needed. Genomic comparisons of 203 A. baumannii strains, collected as part of a surveillance study at the University of Maryland Medical Center, were performed using Large-Scale BLAST Score Ratio (LS-BSR) analysis. For these genomic comparisons, the genomes were grouped according to the date and source of the strain, as well as the carbapenem resistance status. This analysis resulted in the identification of genes unique to specific phylogenomic group, site of isolation, or resistance phenotypes. In addition, this work included identification of genes exclusive to other Acinetobacter species, which may be useful in the future to positively identify A. baumannii as this is currently a difficult clinical task. Phenotypic characterization of the A. baumannii strains resulted in the identification of a phylogenetic cluster of strains that exhibited robust biofilm formation. Genomic analysis of these strains revealed several genes unique to this group and potentially associated with biofilm formation. Mutagenesis of three of these genes was performed and the ability to form robust biofilms was determined. In particular, mutagenesis of a putative pilus assembly gene resulted in significantly decreased biofilm formation, suggesting that this gene plays a key role in the robust biofilm phenotype. In addition, a transcriptional regulator was identified that may play a role in the regulation of genes associated with biofilm formation. Overall, the work presented herein has broadened our understanding of the vast diversity among surveillance isolates of A. baumannii in a single healthcare setting, and has demonstrated the utility of genotypic/phenotypic correlations to identify novel virulence factors.