Vibrio species are zoonotic pathogens that can affect humans by different routes such as ingestion or contact causing gastrointestinal diseases. This study aimed to detect V.cholera and V.parahaemolyticus in Nile Tilapia fish collected from Aswan Governorate, Egypt. A total of 52 muscle samples were collected from Tilapia fish and were subjected to microbiological and molecular characterization. Out of 52 fish muscle samples (Nile Tilapia) 32 appeared as yellow colonies, 2 samples showed green colonies and 18 samples showed mixed yellow and green on TCBS agar. Only 40 samples show biochemically positive for Vibrio species. 15 random samples were amplified to 16srRNA gene by PCR technique for more accurate identification resulting in 14 isolates being positive to 16srRNA, 8 isolates positive for sodB gene-specific of V.cholera, and 6 isolates positive for toxR gene-specific of V.parahaemolyticus. Out of 9 V.cholera isolates were found high resistance rate to amoxiclav (88.8%), gentamicin (77.7%), chloramphenicol (44.4%) and ampicillin (44.4%). Conversely, (55.5%) sensitive to tetracycline, ciprofloxacin and, trimethoprim-sulphamethoxazole, and the average MRA of V.cholera was 0.48. On the other hand, all 6 V.parahaemolyticus isolates were resistant to amoxiclav (100%), tetracycline (100%), gentamicin (100%) and chloramphenicol (100%) However, all isolates sensitive to trimethoprim-sulphamethazole (100%) but half of them sensitive to ciprofloxacin (50%) and ampicillin (50%), and the average MRA of V.parahaemolyticus was 0.59. In conclusion, this study showed that V.cholera is the most dominant pathogenic one then followed by V. parahaemolyticus in Nile tilapia fish resulting in economic losses and causing public health problems.