Hemorrhagic septicemia disease caused by Aeromonas hydrophila bacteria appears to be more common and causes severe damage to many fish species worldwide. This research aimed to isolate, identify, and determine the pathogenicity and antibiotic susceptibility of A. hydrophila in septicemic clown knife (Chitala chitala) fish in the Mekong Delta, Vietnam. As a result, 116 bacterial strains were isolated from 176 septicemic fish samples. Two strains of bacteria, D2F71 and H1F39, were identified as A. hydrophila based on colony morphology, identification results by the API 20E kit, PCR technique, and aerolysin gene sequencing. In addition, these two strains of bacteria were experimentally infected on healthy clown knife fish (weight 15±4 g/fish) by injection method. The experimental results fulfilled Koch's postulates: infected fish showed signs of disease symptoms similar to those of naturally diseased fish. In particular, the two strains of bacteria D2F71 and H1F39 in the experiment had relatively high virulence, with LD50 values of 1.26× 104 CFU/mL and 4.06× 103 CFU/mL, respectively. The obtained findings demonstrated that the bacteria were susceptible to doxycycline, florfenicol, cefotaxime, flumequine, enrofloxacin, and sulfamethoxazole-trimethoprim at a rate of over 70%. On the contrary, the study found that bacterial isolates were resistant to ampicillin (100%), cefazolin (85%), streptomycin (80%), and colistin (80%). To our knowledge, this is the first report on hemorrhagic septicemic clown knife fish caused by A. hydrophila in Vietnam.