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Background and Aim: Otitis media (OM) or middle ear inflammation is an inflammatory disease caused by a bacterial infection such as Pseudomonas aeruginosa (P. aeruginosa), provided with genetic virulence factors. This study aims to detect nan1 and tox A genes of P. aeruginosa isolated from chronic OM patients. Materials and Methods: One hundred thirty OM patients were recruited from Al-Sader medical city and Al-Hakim general hospital in Najaf province from September to January 2021 to collect ear swaps. The Primary identification depends on Gram stain and biochemical tests. We also used Vitek 2 system to make another identification and examine resistance to antibiotics. A polymerase chain reaction (PCR) was used to amplify the required genes. Furthermore, gel electrophoresis was performed to show the amplified genes. Results: The results have revealed that 115 samples (88.46%) give a positive result, while 15 samples (11.54%) are negative for culture bacteria. Only 49 (42.76 %) of the 115 clinical specimens showed positive tests for P. aeruginosa. However, the toxA gene is found in 67.3% of P. aeruginosa isolates, while the nan1 gene is found in 38.77 % of the isolates. Conclusion: Our findings suggest that P. aeruginosa is one of the most common causes of OM infection in Iraqi patients. Most bacterial isolates show the presence of nan1 and tox A genes, with a high prevalence of tox A than nan1. The presence of these genes is one of the virulence factors and probably partly explains the resistance to antibiotics. [ABSTRACT FROM AUTHOR]