Pseudomonas aeruginosa is an opportunistic pathogen causing serious nosocomial infections in patients. Emergence of multi-drug resistant P. aeruginosa is an increasing infection control problem leading to high morbidity and mortality. Extended spectrum beta-lactamase enzymes are the increasing cause of resistance to penicillin’s, cephalosporins, and aztreonam antibiotics in P. aeruginosa. The objective of the study was to determine the prevalence of Pseudomonas aeruginosa from infected patients, antibiotic resistance and occurrence of ESBL producing P. aeruginosa among these isolates. A total of 200 specimens were received by the pathology laboratory of Pakistan Institute of Medical Sciences, Islamabad, Pakistan, which comprised of 50 tracheal 50 pus, 25 bloods, and 25 urine and 50 miscellaneous samples including sputum, swab, wounds, tissue and different body fluids. P.aeruginosa was tested against a panel of 14 antibiotics. The highest percentage of resistance to antibiotics amoxicillin+clavulanicacid, cefoperazone+sulbactum, ceftriaxone,ceftazidime, Piperacillin and tobramycin was measured. The most effective drug established were polymixine B, Nalidixic acid, meropenem, amikacin, imipenem, azetrainum were found as more effective in the order respectively. Among all 200 isolates, 150 were found to be ESBL positive and 50 were ESBL negative. Different factors like gender, age, were also related along with the patient stay in hospital. More males than females were infected having high percentage of Pseudomonas aeruginosa and highest frequency was observed in age group less than 15, gradually declined with increase in age. Since treatment proved to be difficult, prevention is considered as an appropriate means of overcoming infection. Routine detection of ESBLs and careful in vitro testing before antibiotic use may help in the prevention and treatment of patients infected with ESBL producing P. aeruginosa.