Despite the reason that genus Acinetobacter works as a grave human pathogen, very few numbers of researches have been done so that term it as a pathogen in respect to fish. As per the current study, isolation of three pathogenic bacterial strains was carried out from infected blunt snout bream (Megalobrama amblycephala Yih), from a farm in Yixing city, Jiangsu province, China, which displayed symptoms like tail-rot, shedding scales and ascites in addition to gentle ulceration on the entire body regardless of size and sex of fish. Taking into account the bases of morphology, varied biochemical tests, 16S rDNA segment and rpoB gene sequence analysis, in addition to phylogenetic study, the pathogenic bacteria was identified as A. pittii. Recursive infectivity experiment validated their pathogenicity. Pathological modifications of blunt snout bream infected with A. pittii were taken into observation. Confirmation of the pathogenicity was additionally made by infectivity studies of zebra fish (Brachydanio rerio) and nematode (Caenorhabditis elegans). The drug resistance of these isolates was also scrutinized. All isolates, recognized as multiple drug resistant strains, showcased resistance to clindamycin, streptomycin, vancomycin, cephalosporins, ampicillin, piperacillin, and trimethoprim-sulfamethoxazole, while showcasing sensitivity to norfloxacin, gentamicin, amikacin, and imipenem. Multi-locus sequence typing of these A. pittii isolates brought to light a new clonal lineage of Acinetobacter leading to fish septicemia outbreaks together with indicating that Acinetobacter stains with the new sequence type 839 may be the dominant clone. This is the first report dealing with the infection caused by A. pittii in fish that suggests that A. pittii has a prospective threat to be encountered by freshwater fish farming in addition to causing human clinical infections.