BACKGROUND Acinetobacter calcoaceticus baumannii (ACB) complex are Gram-negative opportunistic bacteria with low virulence properties. Their resistance to antibiotics has become a matter of concern in hospital infections. OBJECTIVE The present study aimed to determine the prevalence and antimicrobial susceptibility of ACB isolates collected from the Nemazee hospital of Shiraz. In addition, Pulsed Field Gel Electrophoresis (PFGE) was used to determine the genetic patterns of these strains. METHODS In this cross-sectional study, 93 strains of ACB complex were isolated from patients of Nemazee hospital, Shiraz, Iran. The antibiotic susceptibility patterns of the isolates to the following 15 antibiotics were determined: gentamicin, ticarcillin, ceftazidime, co-trimoxazole, imipenem, piperacillin tazobactam, amikacin, aztreonam, sulbactam, meropenem, tobramycin, cefotaxime, ceftriaxone, colistin, polymyxin B. Pulsed Field Gel Electrophoresis was used to determine the clonal relationship of these strains. RESULTS Most of the isolates were found to be resistant to cefotaxime, co-trimoxazole, ceftriaxone, aztreonam, ceftazidime and ticarcillin (90%), and the least resistance was observed to colistin and polymyxin B. Among the 93 tested samples, 35 antimicrobial susceptibility patterns and 47 PFGE patterns were obtained. CONCLUSIONS High resistance to antibiotics was observed among the strains of ACB complex and the least resistance was towards colistin and polymyxin B, indicating that these antibiotics could be effective for treatment, in case there is no other choice. Using PFGE, the similarity between some strains of Acinetobacter was determined, which indicated epidemics in different parts of the hospital; such epidemics can in turn lead to increased incidence of Acinetobacter infections.