| In order to determine the cause of the large number of deaths of the sick American eel (Anguilla rostrate) in an aquaculture fish farm in Haikou (Hainan), one dominant strain (named AB01) was isolated from the liver and spleen of the diseased American eels. The dominant strain AB01 was identified as Acinetobacter baumannii based on the results form physiological and biochemical testing, sequence alignment of 16S rDNA gene and phylogenetic analysis. The challenge test in healthy American eels by artificial reentry indicated that A. baumannii was the pathogenic bacteria. Further analysis of pathogenicity showed that the semi-lethal concentration of A. baumannii (AB01) was 4.63×106 CFU/mL. Histopathological observation of liver from diseased Anguilla anguilla showed the arrangement of some hepatic cords was disordered, and the cells were swollen, together with dissolved nucleus, loose cytoplasm, and fuzzy structure. Moreover, a lot of vacuoles of different sizes were present in liver. In addition, spleen from diseased Anguilla rostrata presented sparse cytoplasm and extensive injury. The results of antimicrobial susceptibility test showed that A. baumannii (AB01) was resistant to many antibiotics, but was highly sensitive to cephalothin, cefazolin, azithromycin, amikacin, enoxacin, rifampicin, ciprofloxacin, ofloxacin, and medium sensitivity to nine antibiotics, such as florfenicol and so on. Moreover, A. baumannii (AB01) was resistant to twenty antibiotics, such as aztreonam and so on. Thus, this study provides a scientific support for the prevention and control of the disease of the American eel.