Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a model bacterium for studying virulence and bacterial social traits. While it can be isolated in low numbers from a wide variety of environments including soil and water, it can readily be found in almost any human/animal-impacted environment. It is a major cause of illness and death in humans with immunosuppressive and chronic conditions, and infections in these patients are difficult to treat due to a number of antibiotic resistance mechanisms and the organism’s propensity to form multicellular biofilms. One hundred twenty clinical samples and forty hospital environmental samples (various sources) were collected from hospitals in Baghdad city during the period from October 2020 to January 2021, Cetrimide agar, the differential medium was used to isolation and diagnosis of Pseudomonas aeruginosa and 76 (63.33%) clinical isolates and 8 (20%) environmental samples were able to grow, and diagnosed as P. aeruginosa using the conventional biochemical tests, the Api 20 system and 16S rRNA gene. The antibiotic susceptibility test for fourteen antibiotics was performed by the standard disk diffusion method; the results showed that the bacteria were resistant to the most of the antibiotics used in this study and have the ability to form the biofilm.