Background: Although many studies have investigated the mechanisms of cholesteatoma formation, its pathogenesis remains unclear. The aim of this study was to evaluate the levels of periostin, fibronectin and tenascin-C, which are involved in many mechanisms, such as cell adhesion, cell differentiation, inflammation, fibrosis, angiogenesis and cell proliferation, in patients with chronic otitis media (COM). A total of 80 participants, 65 COM patients undergoing surgery and 15 healthcare personnel volunteers serving as controls, were included in this study. The participants were divided into four groups: cholesteatoma, granulation, avivation and control group. The serum periostin, fibronectin and tenascin-C levels of all participants were determined biochemically. Histopathological evaluation of tissue samples and 20 skin samples used as controls was performed by immunohistochemistry. Results: Of the 65 patients, 22 presented with cholesteatoma, 15 with granulation tissue and 28 with the edge of the tympanic membrane perforation freshening tissue. There were no significant differences in serum periostin, fibronectin and tenascin-C levels between the groups. In the immunohistochemical evaluation, fibronectin and periostin staining was significantly more intense in the cholesteatoma group than in the other groups (p = 0.001). Epithelial tenascin-C staining was significantly more intense in the avivation group than in the other groups (p = 0.041). Conclusion: The levels of periostin and fibronectin were higher in cholesteatoma tissue than in other forms of chronic otitis and skin tissue. This suggests that they may be involved in the mechanism of cholesteatoma formation. These proteins could be used as biomarkers in the diagnosis and treatment of cholesteatoma.