Objective: It is recognized that the various types of otitis media (OM) follow a continuum with early forms of the disease leading to more chronic forms over time. This study investigates if biofilms play a role in conjunction with the continuum of pathological changes in different stages of OM. Method: Thirty human temporal bones of each type, sero- purulent OM (SPOM), purulent OM (POM), chronic OM with purulent effusion (PCOM), and fourteen mucoid OM (MOM), were selected. We defined “web-like structures” with host defense cells and bacterial bodies as biofilm. We examined the occurrence, frequency, and anatomic locations of biofilms. Results: Biofilms were observed in 10 (33.3%) of the 30 temporal bones with SPOM, 19 (63.3%) with POM, and 20 (66.7%) with PCOM. A significant difference of biofilms’ frequency between SPOM and POM ( P = .038), and SPOM and PCOM ( P = .017), was observed. The most common anatomic location was mastoid antrum and mastoid cells (35 of 49 temporal bones, 71.4%). The number of anatomic locations with biofilms was 2.20 ± 1.03 in SPOM, 2.89 ± 1.70 in POM, and 4.15 ± 1.60 in PCOM. There was a significant difference of locations between PCOM and SPOM ( P < .001), and PCOM and POM ( P = .022). Conclusion: The frequency of biofilms suggests that the presence of biofilms is associated with the severity of OM. The growth and development of biofilms may play an important role in the progression of chronicity of OM.