The aim of this single-arm prospective study was to determine the flora of the external auditory canal (EAC) in inactive chronic otitis media and evaluate the alteration of microorganisms of the EAC during tympanoplasty upon povidone-iodine antisepsis. Sixty-three patients with central tympanic membrane perforation were enrolled in the study. Preoperative swab cultures were obtained and the EAC was packed with povidone-iodine absorbed gauze. Type I tympanoplasty via a retroauricular route was performed. Cultures from the EAC were taken at the end of each operation. Isolated organisms were identified based upon microbiological, morphological, and biochemical characteristics. The most commonly isolated organisms from preoperative samples were normal commensal flora, including 73 coagulase-negative staphylococci (CNS) and 18 diphtheroid bacilli (DB). Less commonly cultured pathogenic species included four isolates of Staphylococcus aureus and three isolates of Candida albicans. No bacteria were observed in five patients. Following povidone-iodine antisepsis, 32 of the samples were negative. Eradication was statistically significant for CNS, DB and pathogen microorganism (P < 0.05). Isolated bacteria differed from those in preoperative swab cultures in eight cases. After antisepsis, diverse strains of the CNS were isolated in 13 cases and 10 patients showed no change in microbial flora. Postoperative culture demonstrated that all seven pathogenic isolates were eradicated (100 %); this selective efficacy of povidone-iodine antisepsis against pathogenic isolates was significant when compared with commensal flora (P < 0.05). These results suggest that povidone-iodine antisepsis of the EAC before tympanoplasty is an effective method for the elimination microorganisms, especially pathogenic bacteria.