AbstractChronic otitis media is a common middle ear disease in otolaryngology and head and neck surgery. Bacterial infection is considered the main cause of disease, but relying on conventional bacterial cultures can be problematic for identification of specific pathogens. Current research suggests that bacteria in microbial communities can only be identified by rDNA sequencing of bacteria.This prospective study utilized broad-range PCR amplification of 16S rRNA genes with clone analysis to compare bacterial diversity in lesions from 6 patients with chronic suppurative otitis media (CSOM) and 10 patients with cholesteatoma of middle ear lesions. Bacteria were analyzed at the phylum, order, family, genus, and species levels. Bacterial species abundance and species diversity were greater in cholesteatoma of the middle ear lesions than in CSOM lesions. At all bacterial taxonomic levels, the epithelial tissue of middle ear cholesteatoma was complex in terms of bacterial diversity, covering a large number of Gram-positive and Gram-negative bacteria, likely related to bacterial microbiome formation. In contrast, bacteriology of the CSOM lesions was relatively simple at all taxonomic levels, with all sequences characterized as belonging to Gram-negative bacteria. These results suggest that persistent infection of middle ear cholesteatoma may be a microbial flora disorder, which is related to conditional pathogenic bacteria, rather than a single bacterial infectious disease. Findings from the study may have implications in the selection of antimicrobial agents for the treatment of chronic otitis media.