Nile tilapia (Oreochromis niloticus) aquaculture represents one of the most important cultivation species in Egypt. However, Tilapia farming encounters some problems that result in high fish mortalities and huge economic losses due to bacterial pathogens. Thus, the current study aimed to isolate, identify, and characterize the pathogenic bacteria affecting Nile tilapia farming in El-Abbassa, Abo-Hammad, Sharkia. Besides, antibiotic susceptibility to pathogens was determined as a primary step for controlling diseases.  Hence, a collection of 252 fish samples was gathered from 200 fish farms. Upon microbiological and biochemical analysis, the presence of only 3 bacterial genera was detected, including Gram-negative bacteria of Aeromonas spp. (52.80 %), Pseudomonas spp. (27.9 %), and Vibrio spp. (19.3 %). Aeromonas sobria showed the highest prevalence in the infected Oreochromis niloticus. Antibiogram test revealed high levels of resistance expressed by all bacterial isolates to Ampicillin, Amoxicillin, and Erythromycin. On the other hand, Norfloxacin was effective against all isolated bacteria followed by Ciprofloxacin. Establishing effective control methods for pathogenic bacteria would greatly enhance fish production.