Whereas it is well documented that the genus Acinetobacter is associated with antibiotic resistant bacteria and human clinical infections, there are very few studies to date that report it as an emerging new pathogen for fish. In the present study, Acinetobacter lwoffii was isolated from diseased rainbow trout (Oncorhynchus mykiss) from Iranian farms, in the Khuzestan Province by both biochemical tests and polymerase chain reaction (PCR)-based methods and further confirmed as the causative agent by infectivity experiment. Uni or bilateral exophthalmia, blackening of the skin, abdominal distension, hemorrhages around the mouth cavity and eyes, basal fine, skin, gills and in internal organs were the main clinical signs in the affected fish. Phylogenetic analysis of the 16S rRNA sequence of MD77 revealed a 99% homology with that of A. lwoffii (GenBank accession no.KR856323.1) form Iran. Histopathological changes of challenged rainbow trout including focal necrosis of liver cells (hepatocyte), pyknotic nucleus and karyolysis of hepatocyte, hyperemia, fatty acid composition change of liver, infiltration of inflammatory cells, sinusoidal dilatation and congestion. The drug resistance of isolates and minimal inhibitory concentration (MIC) was examined and showed that A. lwoffii was multiresistant to 8 of the antimicrobial agents tested..