Background and Objectives: Non-fementer gram-negative bacilli (NFGB) are ubiquitous pathogen that has emerged as a major cause of health care associated infections. The aim of this study was to determine the prevalence and antimicrobial susceptibility of NFGB in an Iranian hospital. Materials and Methods: FromJuly 2005 to November 2006 a total of 257 strains of NFGB including 109 (42.41%) strains of Pseudomonas aeruoginosa, 88 (34.24%) strains of Acinetobacter baumannii, 48 (18.67%) stains of Stenotrophomonas maltophilia and 12 (4.66%) strains of Burkholderia cepacia were isolated from clinical specimens taken from patients hospitalized in an Iranian 1000–bed tertiary care hospital[d1] . Conventional bacteriological methods were used for identification and susceptibility testing of NFGB. Susceptibility testing was performed by method as recommended by Clinical Laboratory Standard Institute (CLSI). Data were analyzed using SPSS 11.5 for Windows (SPSS Inc., Chicago, IL) Results: A total of 257 non-duplicating of NFGB strains were isolated from 234 hospitalized patients. The most effective antibiotic against P. aeruginosa and A. baumannii was imipenem followed by tobramycin. Fluoroquinolones had moderate activity against P. aerugunosa. Most isolates of A.baumannii were multi-drug resistant. Susceptibility of S. maltophila to ticarcillin- clavuanic, ofloxacin and ceftazidim was 96%, 94% and 81%, respectively. Thirty three percent of this bacterium isolates were resistant to co-trimoxazole. Conclusion: In our study, imipenem was the most effective antibiotic against P. aeruginosa and A. baummannii isolates. Previous history use of antibiotics, longer duration of hospital stay and mechanical ventilation were the major risk factors for resistance acquisition in NFGB especially in P. aeruginosa and A. baumannii.