Pseudomonas aeruginosa has the ability to adapt to many situations. So, from El-Temsah Lake and fish farms in Ismailia governorate, samples were taken from fish that exhibited signs of septicemia and others that appeared to be healthy without any symptoms. Further, about 60 human samples were taken from patients at the Suez Canal University Hospital in Ismailia governorate, as well as 23 water samples from El-Temsah Lake for P. aeruginosa isolation and PCR confirmation. The bacteriological analysis revealed that P. aeruginosa was found in 33.4 % of all analyzed samples from various sources (Fish, human and water). By traditional cultivation, the prevalence of P. aeruginosa was 34.33 % in fish samples, 28.3 % in human samples, and 21 % in water samples. The PCR assay was used to validate P. aeruginosa isolates from various sources in a quick and precise manner. Genetic confirmation of 15 biochemically determined P. aeruginosa isolates was done using the 16S rDNA sequence with a particular band at 956 base pair. This study proved that P. aeruginosa is widely distributed in all sources with different rates and it has a public health significance.