Background: Chronic otitis media (COM) of mucosal type is a chronic condition of middle ear characterised by permanent perforation of tympanic membrane and hearing impairment, which is commonly seen in developing countries due to various factors such as low socioeconomic status, virulence of the organism, poor personal hygiene, increased antibiotic resistance, recurrent episodes of upper respiratory tract infections and inadequate health facilities. So, early and effective measures to be undertaken to prevent possible complications. Aim: To determine whether there is any difference in bacterial isolates in clinically diagnosed chronic otitis media compared with adenosine deaminase enzyme (ADA) level proved chronic otitis media. Materials and methods: Total 80 patients were included in the study selected on a random basis and divided into two groups. Group A consisted of 40 patients diagnosed clinically as chronic otitis media and group B consisted of 40 patients diagnosed as ADA level proved chronic otitis media. The discharge was collected from the middle ear with a sterile cotton swab and sent to the microbiology department immediately for further processing of the sample and to identify the microorganisms. A blood sample is collected from 40 patients of group B to estimate the adenosine deaminase level. Results: We have deciphered that Pseudomonas aeruginosa was the most common bacteria isolated in both the groups. In this study, none of the samples shows mixed growth or anaerobic organisms. Conclusion: From this study, we concluded that the increased level of serum adenosine deaminase can be used as one of the indicative tools in chronic otitis media and its raise indicates the chronicity of the disease. ADA level will be an essential diagnostic tool in identifying the bacterial isolates.