Nosocomial infection is an increasing problem. The global problem of antimicrobial resistance is particularly pressing in developing countries, where the infectious disease burden is high and cost constraints prevent the widespread application of newer, more expensive agents. In a prospective study, 203 consecutive cases with hospital-acquired infection in a university hospital in Bushehr port were evaluated. The most common hospital-acquired infection was urinary (76 cases), conjunctivitis (16 cases), bacteremia (8 cases), meningitis (5 cases), wound (3 cases), empyema (2 cases) and peritonitis (1 case). The patients with hospital-acquired infection were from surgical and internal medicine I.C.Us (53.2% & 15.6%, respectively). The most frequent isolated organisms were Pseudomonas aeruginosa (25.6%), Acinetobacter baumannii (19.7%), E. coli (13.3%), Klebsiella pneumoniae (11.3%), Staphylococcus aureus (8.4%), Staphylococcus epidermidis (7.9%), Enterobacter species (7%), Streptococcus species (6.4%), and Proteus mirabilis (0.5%). The most resistant organisms to antimicrobial agents were Acinetobacter baumannii and Pseudomonas aeruginosa 97 & 93.3% of these bacteria were resistant to third generation cephalosporins. The isolated Staphylococcal species were resistant to amikacin (94%). In conclusion, gram negative bacteria were the most common etiologic agent of hospital-acquired infection and had a high level of resistance to amikacin and third generation cephalosporins. Therefore, new therapeutic strategies should be designed to combat these microorganisms.