Introduction:Otitis external infection is an inflammation of the ear canal frequently caused by Pseudomonas aeruginosa, followed by Staphylococcus epidermis and Staphylococcus auerus. Objective:This study investigated the spectrum of bacterial and fungal agents that cause otitis external infection in Jordanian patients with an emphasis on important antimicrobial resistance genes and putative virulence factors of P. aeruginosa isolates using molecular PCR methods. Methods:A total of 128 ear swab samples were obtained from outpatients with otitis external infection of Ear-Nose-Throat Clinic (ENT) from the Jordan University Hospital (JUH). All samples were cultured for bacteria and fungi and their growth was identified by macroscopic and microscopic examination as well as recommended biochemical tests. Results:Positive growth of bacteria and fungi were found in 105/128 (82%) of the examined cases. A total of 28 (22%) of the recovered organisms from ear samples were P. aeruginosa. A total of 11/28 (39%) of P. aeruginosa isolates were Multidrug-Resistant (MDR) which are resistant to three or more antibiotic classes. Both blaIMP-15 and VIM genes were not detected, while KPC genes were found in 57% among all isolates. The rates of the potential virulence genes found among 28 P. aeruginosa isolates were as follows: lasB, algD, toxA, exoU PilB and exoS at 100%, 100%, 82%, 72%, 54% and 25%, respectively. All isolates produced beta hemolysis on both human and sheep blood agar and showed either the pigment pyoverdin (57.1%) or pyocyanin (42.8%). Conclusion:Accurate identification of the causative agent of otitis external infection and its susceptibility to antibiotics especially P.aeruginosa is highly important for successful treatment. No significant relationship has been found between MDR P. aeruginosa and the presence of virulence genes.