- Background: Data on the outcome of tympanic membrane (TM) closure after tympanocentesis in acute otitis media (AOM) patients is limited. Objectives: To analyze the dynamics of TM perforation closure after 1 or 2 tympanocentesis procedures performed at diagnosis and during AOM treatment. Patients and Methods: Study population included 113 children enrolled in 4 double-tympanocentesis studies. Only the files of patients whose first and second examination were performed by the same 2 otorhinolaryngologists were analyzed. Middle ear fluid (MEF) was cultured on day 1 and days 4–6, the latter only in initially culture-positive patients. Patients were also examined on days 11–14 and followed until days 22–28. Results: Ninety-three (82%) patients underwent tympanocentesis on days 4–6; 103 (91%) and 95 (84%) were evaluable on days 11–14 and 22–28, respectively. One hundred seventy-three ears underwent tympanocentesis on day 1 and 139 on days 4–6. Ninety-seven (86%) patients had positive MEF cultures. One hundred fifty-three (88%) tympanocentesis procedures performed at enrollment were closed on days 4–6. No differences were recorded in the closure rates as function of patient age, previous AOM history, MEF culture positivity, pathogens isolated at enrollment, and pathogen eradication on days 4–6. The 20 eardrums still open on days 4–6 were closed on days 11–14. Eleven (9%) of the evaluable ears tapped on days 4–6 were not closed on days 11–14 and 9 of 10 were closed on days 22–28. Conclusions: TM perforation closed in most cases within a few days regardless of patient and disease characteristics, and 2 consecutive tympanocentesis procedures performed at short-time intervals are associated with good TM closure rates.