Acinetobacter species are gram negative non fermenters, which are important nosocomial pathogens involved in various outbreaks in hospitals due to widespread resistance to majority antibiotics. The aim of this study is to speciate Acinetobacter isolated from clinical samples, to assess the antibiotic sensitivity pattern and to detect the production of metallo-β-lactamase by double disc synergy test. The study was conducted in the department of microbiology, A.J Institute of Medical Sciences. All clinical samples were subjected to gram stain & cultured; the Acinetobacter isolates obtained were subjected to antibiogram. Those isolates that showed Imipenem resistant were further tested for production of metallo-β-lactamase by double disc synergy test. Out of 6625 culture positive isolates, 414 (36.1%) were identified biochemically to belong to Acinetobacter species. Of the 414 cases, 393 (94.9%) were further identified to be Acinetobacter baumannii and the remaining 21 (5.1%) to be Acinetobacter lwoffi. Acinetobacter lwoffii showed 100% sensitivity to all the drugs. Of the 393 Acinetobacter baumannii isolates 109(27.7%) showed resistant to Imipenem. Out of these 109 isolates, 65 (59.63%) were positive for metallo-β-lactamase production by double disk synergy test. The speciation is highly demanding and laborious but it’s important to be demonstrated due to difference in the antibiotic susceptibility pattern. Carbapenem resistant Acinetobacter nosocomial strains in ICUs are detected to be more resistant to antibiotics. As shown in this study the metallo-β-lactamase producing A.baumannii isolates were 59.63% and therapeutic options were limited. Therefore early identification of metallo-β-lactamase producers is of great importance to start appropriate treatment and to control the spread.