Biofilm formation is present in the middle-ear mucosa of chronic otitis media(COM) patients andCOMis a biofilm-related disease. Biofilms are organized and complex communities in which bacteria communicate to each other and gain tremendous advantages. In this unique structure, bacteria can diffuse nutrients, gain resistance to antimicrobials agents and host defense mechanisms. Recently bacterial biofilms have been proven to be important in infectious diseases of head and neck region. Aprospective case-control study was conducted. The study group comprised of patients with chronic otitis media and patients undergoing surgery for cochlear implantation was involved in the control group. Study group also divided to subgroups SSA and SSB according to history of ear discharge within last six months. Direct microscopy (DM) and transmission electron microscopy (TEM) were used to assess presence of biofilms. Totally 19 patients, 10 with ear discharge history within last 6 months and 9 without discharge comprised the study group. Control group comprised of 9 patients undergone cochlear implantation. In all of the patients with ear discharge history and in two of the patients without ear discharge history, biofilm formation was detected by both DM and TEM. All control group members were free of biofilm formation. The differences were statistically significant between study and control groups (p = 0.002) and between study subgroups (p < 0.001); but not significant between study subgroup without ear discharge history and control group (p = 0.470). In the middle ear mucosa of patients with chronic otitis media, biofilm formation is common, especially when ear discharge history is present.