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This work aimed to investigate the virulence factors and antibiotic resistance of A. hydrophila isolated from different sources in Damietta Governorate, Egypt. The samples comprised of tilapia (Oreochromis niloticus, n=150), mugil (Mugil cephalus, n=144), human stool (n=102) and fish sellers hand swabs (n=85). A total of 25 A. hydrophila isolates were recovered and molecularly confirmed, of which, 2.7% were from tilapia muscles, 2% from tilapia viscera, 6.3% from mugil viscera and 8.8% from stool samples. No isolates were recovered from mugil muscles and hand swabs. Phenotypic determination of heamolysis, lipolysis, proteolysis and gelatinase activity revealed that 60%, 56%, 60% and 100% were positive, respectively. Molecular identification of haemolysin (hly), aerolysin (aer), lipase (lip) and cytotoxic enterotoxins (act) virulence associated genes showed that 28%, 68%, 28% and 16% of the examined isolates were positive, respectively. Antimicrobial susceptibility of the isolates to 17 antibiotics was determined, the most resistance pattern was observed for cefixime (88%), while, all of the isolates were susceptible to imipenem. This study revealed that A. hydrophila isolated from fish and humans showed several virulence factors and exhibit a wide range of antibiotic resistance which is considered a public health hazard as well as.