Acinetobacter spp. are the frequent causes of nosocomial infections which are difficult to treat due to multidrug resistance. The aim of this study was to determine the antibiotic susceptibilities and the presence of metallo-beta-lactamases in Acinetobacter spp. isolated from patients admitted to Hacettepe University Adult Hospital. A total of 124 Acinetobacter spp. isolates were included in the study. Antibiotic susceptibilities against imipenem (IMP), meropenem (MER), ceftazidime (CAZ), ciprofloxacin (CIP) and aztreonam (AZT) were studied by microdilution susceptibility testing according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Multidrug-resistant isolates (MDR) were further tested for susceptibility against Colistin by microdilution, and against amikacin (AN), piperacillin-tazobactam (PIP-TAZ), cefepime (FEP), ceftriaxone (CRO), tetracycline (TET), trimetoprim- sulfomethoxazole (SXT) and mezlocillin (MEZ) by disk diffusion method according to CLSI guidelines. Each isolate was also tested for metallo-beta-lactamase (MBL) production by using IMP and EDTA combined disk diffusion test and molecular analysis for blaIMP-1 and blaVIM-2 genes was done by polymerase chain reaction (PCR). Among 124 non-duplicate isolates, 72 were identified as Acinetobacter baumannii and 52 as Acinetobacter lwoffii. Minimum inhibitor concentration 50 (MIC50) and minimum inhibitor concentration 90 (MIC90) values of the isolates were 32 and 128 μg/ml for IMP, 16 and 32 μg/ml for MER, 128 and 256 μg/ml for CIP, 64 and 256 ug/ml for CAZ, 128 and 256 μg/ml for AZT, respectively. Forty-three (34.7%) isolates were susceptible to IMP. Overall, 51 (41%) Acinetobacter spp. were found to be resistant to ≥ 3 antibiotics belonging to different antimicrobial classes and defined as MDR. Colistin MIC50 and MIC 90 values were 2 and 8 μg/ml, respectively and the rate of Colistin resistance was 27.5% in MDR isolates. The resistance rates for AN, PIP-TAZ, FEP, CRO, TET, SXT and MEZ were 80.4%, 98%, 92.2%, 100%, 100%, 86.3% and 86.3%, respectively. Among 124 isolates, 64 (51.6%) yielded positive result by IMP-EDTA combined disk test, and all the isolates were negative in terms of the tested MBL genes by PCR. These data revealed high level resistance among the Acinetobacter population in our hospital. The high rate of carbapenem resistance and increasing Colistin resistance among Acinetobacter isolates should be surveyed cautiously. The increasing Incidence of multidrug resistant Acinetobacter spp. emphasizes the need for new and effective treatment options.