The aims of this study were isolation and characterization of Vibrio species isolated from fish meat. A total 100 fish samples including chilled Tilapia nilotica, chilled cat fish (Clarias gariepinus), frozen mackerel and frozen shrimp were collected from various markets in Sohag governorate. Vibrio species were isolated from 41% of the examined samples. The prevalence of Vibrio was the highest in chilled Tilapia nilotica (80%) followed by frozen shrimp (40%) then frozen mackerel (32%) finally chilled cat fish was (12%). Seven Vibrio species were identified using the biochemical tests including Vibrio cholerae, Vibrio metschnikovii, Vibrio parahaemolyticus, Vibrio carchariae, Vibrio vulnificus, Vibrio damsel and Vibrio mimicus,. For further confirmation selected isolates were identified by a multiplex PCR by using species-specific primers to amplify gene regions in three species sodB gene for Vibrio cholerae, flaE gene for Vibrio parahaemolyticus and Hsp60 gene for Vibrio vulnificus. All isolates were susceptible to trimethoprim/sulfamethoxazole except Vibrio mimicus Furthermore, the resistance of Vibrio cholera against streptomycin and erythromycin was recorded. Testing the ability of Vibrio for biofilm formation on Congo red plates was resulted in some Vibrio species including Vibrio cholera, Vibrio metschinkovii and Vibrio damsela had the ability to form biofilms. Rosemary essential oil had a great antibacterial activity against all isolated Vibrio species while both oregano and olive oil had no antibacterial activities on Vibrio species. The results of the current work concluded the occurrence of Vibrio species in some examined fish samples and rosemary essential oil is a promising compound for controlling Vibrio.