Hypothesis: We designed a study to determine the role of mastoid mucosal biofilm in chronic otitis media (COM)., Background: Biofilm formation has been found in several chronic airway infections. COM is associated with chronic, recalcitrant infection of the mastoid mucosa, and surgery often is required., Methods: COM patients were divided into 2 groups: one with chronic suppurative otitis media (CSOM) and one with cholesteatoma presence. All COM patients had mastoid involvement in a preoperative computed tomographic scan. The control group consisted of patients undergoing cochlear implantation, with no previous history of chronic otitis media. Mastoid mucosa samples were harvested during mastoidectomy. The samples were studied with multiplex-polymerase chain reaction and with CSLM using BacLight Live/Dead stain. Routine bacterial culture was performed in selected cases., Results: A total of 29 COM patients underwent mastoidectomy. Mastoid mucosal biofilm formation could be found in 19 (66%) of these patients. In the control group, there were 11 cases of cochlear implantation, and 1 patient (9%) presented mastoid mucosal biofilm. In the cholesteatoma group, there were 17 patients, of which, 14 (82%) presented biofilm, whereas in the CSOM group, 5 (42%) of 12 patients presented biofilm. The correlation between COM and biofilm was statistically significant (Fisher's exact test, p = 0.003), as was the correlation between cholesteatoma and biofilm, in comparison with the CSOM group (Fisher's exact test, p = 0.046)., Conclusion: Mastoid mucosal biofilm could be seen in patients with COM with or without cholesteatoma. The role of mastoid biofilm in the development of cholesteatoma should be studied further.