Nile-Tilapia (Oreochromis niloticus) aquaculture represents one of the most important cultivation species in Egypt. However, Tilapia fish farming is challenged by some problems. Of those, the presence of bacterial pathogens resulting in high fish mortalities and huge economic losses. Thus, the current investigation aimed to isolate, identify, and characterize the pathogenic bacteria from Nile tilapia fish farm in El-Abassa village, Egypt and to investigate their antibiotic susceptibility as a primary step for controlling diseases. 182 bacterial isolates were obtained from one hundred Tilapia fish samples. The microbiological and biochemical analysis of the examined fish indicated the presence of only 5 bacterial genera. Three of them are Gram-negative bacteria (representing 86.26% of total isolates) including Aeromonas spp. (46.70 %), Pseudomonas spp. (23.08 %), and Vibrio spp. (16.48 %). While two genera are Gram-positive bacteria (representing 13.74% of the total isolates) including Streptococcus spp. (8.79 %) and Staphylococcus spp. (4.95 %). This indicates that Gram-negative bacteria are the main cause of high fish mortalities in the studied area while Aeromonas hydrophila exhibited the highest prevalence in infected tilapia. Antibiogram test revealed high levels of resistance expressed by all isolates to ampicillin, amoxicillin, and erythromycin. On the other hand, norfloxacin was effective against all isolated bacteria followed by ciprofloxacin; therefore, norfloxacin should be recommended as a supplement in fish fed-diets to control the bacterial infection. Establishing effective control methods for pathogenic isolates would greatly enhance fish production.