A microscope-based optical coherence tomography (OCT) device was used to assess the microanatomy of the tympanic membrane in patients with chronic myringitis. A prospective study was designed for this purpose. OCT measurements of the tympanic membrane were done on 11 patients with myringitis with a microscope-based spectral domain OCT system. The in vivo findings were compared with those findings of a control group consisting of 36 patients with retraction pockets or atrophic tympanic membranes (n = 13), myringosclerosis (n = 12) and perforations (n = 11). In active chronic myringitis, the thickness of the tympanic membrane is increased compared to healthy membranes and to other pathological conditions of the tympanic membrane. Consistent changes of the microanatomy of the tympanic membrane were found in chronic myringitis with OCT. Serial OCT measurements revealed no biofilm suspicious findings in all patients with active chronic myringitis. Intraoperative and in vivo OCT measurements may help to detect microanatomical changes of the tympanic membrane in chronic myringitis and in other conditions of the tympanic membrane.