The current study was carried out to isolate and identify some of bacteria infecting fish of El-Serw fish farm during the harvest season from December to March 2014-2015. Fish samples (155) were collected from different ponds of farm; 65 Nile tilapia (Oreochromis niloticus), 55 catfish (Clarias gariepinus) and 35 mullet (Liza ramada) were used to isolate bacteria in fish organs (gills, liver and kidney) using different selective media, from the examined fish, bacteria belonging to Pseudomonas, Aeromonas and Enterobacteriaceae were isolated. The group most frequently isolated from fish was Aeromonas, the highest number of putative Aeomonas isolates were obtained from O. niloticus 19 (29%),followed C. gariepinus 12 (21.8%) and L. ramada 5 (14.2%). Whereas putative Pseudomonas sp.showed fewer prevalence 9 (13.8%), 8 (14.5%) and 4 (11.4%) from O. niloticus, C. gariepinus and L. ramada, respectively, while only two colonies were recovered from Yersinia Agar Base. Seven isolates were selected and identified by sequencing of 16S rRNA. The analysis of sequence of 16S rRNA gene using universal primers (RW01 and DG74) resulted in the identification of three bacterial isolates of Pseudomonas fluorescens, one of Pseudomonas putida, two of Aeromonas hydrophila and one of Klebsiella oxytoca. The antibiogram test of these isolates revealed their sensitivity to ciprofloxacin, norfloxacin, and gentamycin.  This study concluded that the performance 16S rRNA assay has the prospective to create an important contribution to infection management providing rapid of identification the bacterial pathogens in fish.