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Pseudomonas aeruginosa is one of the common pathogens causing serious infections in fish, consequently high economic losses in freshwater aquaculture. This study was carried out to investigate the prevalence, antibiotic sensitivity, and distribution of virulence genes (oprL, toxA, exoS, fliC and pelA) and antibiotic resistance genes (blaTEM and blaCTX-M) related to natural infection of P.aeurginosa among some cultured freshwater fish. A total of 100 Oreochromis niloticus fish were randomly collected from EL-Manzla lake in Port-Said Governorate, Egypt. Clinical and bacteriological examinations were performed on P. aeruginosa, recovered from O. niloticus (32/100; 32%). The PCR results revealed that all isolates harboured 16srRNA conserved gene, oprL virulence gene, whereas only 87.1%, 84.3% were positive for the toxA and pelA gene, respectively. In addition only 37.5% and 15.7% were positive for the fliC and exoS gene, respectively. Depending on the antibiotic sensitivity and molecular analysis of resistance genes, 12.5% of the tested isolates exhibited multi-drug resistance (MDR) to four antimicrobial classes (penicillins, cephalosporins, tetracyclines and sulfonamides) and harboured the blaTEM and blaCTX-M resistance genes as well as oprL, toxA and pelA virulence genes. MDR to three antimicrobial classes (penicillins, cephalosporins and fluroquinolones) was demonstrated by 9.4% of the tested isolates which harboured the blaTEM and blaCTX-M resistance genes as well as oprL, toxA and pelA virulence genes. Of tested isolates, 12.5% exhibited extensive drug resistance (XDR) to five antimicrobial classes (penicillins, cephalosporins, tetracyclines, aminoglycosides and sulfonamides) harbouring the blaTEM and blaCTX-M resistance genes as well as oprL, toxA, exoS and pelA virulence genes. The emergence of MDR and XDR strains is regarded as a public health issue and indicates improper treatment and a bad prognosis of infections caused by P. aeruginosa [ABSTRACT FROM AUTHOR]