Cymothoid isopods are known to have harmful impacts on fish. Hence, their host-parasite relationship must receive much attention. This study investigated the phenotypic and genotypic characterization of Vibrio alginolyticus recovered from both fish and isopod which may correlate isopod to the transmission of V. alginolyticus. Thirty-two V. alginolyticus strains from both freshly dead moribund fishes and their isopods were studied; 18 strains from Tilapia zilli in Manzla Lake and 14 strains from Solea aegyptiaca in Qaroun Lake. In addition,, 3 strains were isolated from the water of both lakes. Biochemical, antibiogram, species-specific PCR and phylogenetic analysis of 16S rRNA gene were performed. Moreover, water physicochemical factors and isopod identification were done. The physiochemical analysis of water samples from both lakes showed bad water quality of high temperature, low dissolved oxygen levels and high values of almost all chemical parameters. The results revealed the isolation of Nerocila orbignyi isopod in both Manzla and Qaroun Lakes, while Levonica redmanii isopod was only found in Qaroun Lake. V. alginolyticus strains from fish, isopods and water showed the exact antimicrobial susceptibility profile. They were susceptible to erythromycin, imipenem and trimethoprim/sulphamethoxazole, while they were resistant to ampicillin, lincomycin, colistin and penicillin. In the species-specific PCR using the collagenase gene, all tested isolates showed the characteristic V. alginolyticus amplicon of 737 bp. The phylogenetic analysis of partial 16S rRNA gene sequence revealed that V. alginolyticus isolates from fish and isopod showed 100% similarity with each other and 99.23% similarity with water strain, suggesting that the isopod was the likely source of infections. In conclusion, we demonstrated that the deterioration of water quality strongly related to the high prevalence of isopods could be a potential vector in the transmission of V.alginolyticus in T.zilli and S. aegyptiaca  fish.