Background: Acinetobacter baumannii has emerged as a significant hospital pathogen, quickly becoming resistant to commonly prescribed antimicrobials. Objectives: To isolate various species of Acinetobacter, to compare inpatients (ICU’s & wards) and outpatients isolates and to know it’s frequency from various clinical specimens. Material and methods: The retrospective study is conducted in the department of Microbiology, Krishna Institute of Medical Sciences, Secunderabad, from January 2013 to December 2014. The various clinical specimens from inpatients and outpatients were included. The samples were processed as per the standard guidelines. Identification & antibiotic sensitivity testing was done by using GN and AST 281 cards (Vitek 2 compact, BioMerieux) respectively. MIC values of antibiotics were obtained and reporting was done as per the CLSI guidelines. The data was captured from the laboratory computer and analysed. Results: A total of 496 Acinetobacter species were isolated from 2459 samples (20.17%) from the entire hospital, in which Acinetobacter baumannii was 462(93.16%), Acinetobacter lwoffii was 16(3.22%), Acinetobacter junii was 13(2.62%), Acinetobacter haemolyticus was 5(1.00%). Maximum isolates observed from endotracheal tube secretions (39.51%) followed by blood specimens (15.12%), sputum (12.70%), pus swab (8.66%), clean catch (5.84%) and others (18.17%). Conclusions: In this study, Acinetobacter isolates showed multidrug resistant pattern mostly in inpatients and hence there is a need for emphasizing the importance of hand washing and use of disinfectants in prevention of transmission of infection in health care setups. Keywords: Acinetobacter; multi-drug resistance; Acinetobacter baumannii; Acinetobacter haemolyticus; VITEK