BACKGROUND The middle ear mucosa (MEM) plays a central role in the middle ear due to its function of providing regular ventilation. To date, assessment of the state of the MEM is only possible subjectively by the surgeon. An objective characterization of the state of the MEM is desirable. OBJECTIVE The aim of this study was to enable objective characterization of the MEM and test infrared (IR) spectroscopy as a possible diagnostic tool for clinical use. METHODS During middle ear surgery, 48 MEM samples were collected and divided into four groups according to clinical appearance: group I: normal MEM; group II: sclerotic MEM; group III: inflammatory thickened MEM; group IV: granulated MEM. After collection, samples were analyzed by IR spectroscopy to identify characteristic IR spectra. RESULTS In the supervised analysis of the selected images, the biochemical differences representing the decisive factors for classification into groups I to IV were characterized. The differences in amide bands, carbohydrates, lipids, and proteins permit reliable separation of the clinical categories. CONCLUSIONS Spectroscopic investigations enable objective characterization of the MEM. Conclusions regarding biochemical differences make it possible to weigh up treatment options. Routine use of IR spectroscopy in the operating theater requires histopathological comparison and an extended dataset with reference values of the individual groups.