This study was conducted on 100 diseased Nile tilapia (O. niloticus) fish of various sizes collected from different fish markets in Kaliobia Governorate to estimate the prevalence of Pseudomonas infection and detection of some virulence genes in the isolated P. aeruginosa strains. The results of bacteriological examination revealed that the prevalence of Pseudomonas septicemia with Pseudomonas species isolation was 17.0% (17 \ 100 examined fish). These cases were attributed to P. anguilliseptica; P. aeruginosa and P. fluorescens (14/43.7%; 12/37.5% and 6/18.8%), respectively. In addition, 32 Pseudomonas species were isolated, 11from liver samples (34.4%); 10 from kidney samples (31.2%); 6 from gill samples (18.8%) and 5 from skin samples (15.6%). Moreover, 14 P. anguilliseptica were isolated with an incidence of 35.7%, 28.6%, 21.4% and 14.3% followed by 12 P. aeruginosa 33.3%, 25%,16.7% and 25% respectively; 6 P. fluorescens 33.3%; 50.0%,16.7% and 0.0% from the liver, kidney, gill and skin samples respectively. The in-vitro antimicrobial sensitivity test showed that the isolated Pseudomonas strains were sensitive to gentamycin; enrofloxacin; norfloxacin; ciprofloxacin and florphenicol. Meanwhile; they were intermediate sensitive for doxycycline; sulfa-trimethoprim; oxytetracycline; nalidixic acid and streptomycin. In contrast, they were resistant for cefotaxime; erythromycin; amoxicillin; methicillin; oxacillin and ampicillin. Moreover, the PCR results revealed that, opr L and exo S virulence genes were detected in all six studied strains (100.0%). Meanwhile, phz M virulence gene was detected in 5 out of 6 studied strains (83.3%) and tox A virulence gene was detected in 4 out of 6 studied strains (66.7%) i.e., all studied strains were Ps. aeruginosa and all of them were virulent strains.