Background and Aim: Acinetobacter baumannii is considered to be a re-emerging causative agent of nosocomial infections. There is a significant relation between pathogenicity of this bacterium and the numerous virulence factors. The purpose of this study was to investigate nine virulence factor genes in A. baumannii isolates derived from hospitalized patients. Materials and Methods: A total of 50 A. baumannii isolates were recovered from patients with pneumonia in Imam Khomeini Hospital, Tehran, Iran. Following biochemical and microbiological identification of the bacteria, Multiplex PCR was performed for basD, plD, csuA genes, surA, pbpG, bfmR genes, and bap, ompA genes using specific sets of primers which were specifically designed for this study. The espA was identified separately by a Uniplex PCR assay. All amplified DNA fragments were sequenced for the products’ confirmation. Results: Among the 50 clinical isolates of A. baumannii studied, bfmR and pbpG genes were reported in all samples (100%), bap, plD, surA, and csuA genes were collected from 49 samples (98%), 48 (96%) of these isolates had ompA and basD genes, and espA gene was observed in only five isolates (10%). Conclusion: According to this study results, virulence factors genes in clinical A. baumannii have a prevalence rate more than 90%. Additionally, the high incidence rate of those genes related to biofilm formation indicates that most clinical strains have the ability to form biofilm structures.