© 2021 Elsevier Inc.This study aimed to investigate the bacteriome in microscopically healthy middle ear mucosa using Next-generation sequencing (NGS) technology. A total of 60 middle ear washing fluids of pediatric and adult were obtained from 47 patients (35 children and 12 adults). Both children and adults with normal middle ears harbored diverse bacteriome. Seventeen different genera with a mean relative abundance of more than 1% were detected in all samples. Both in adult and children, the most abundant genus was Propionibacterium followed by Streptococcus, Staphylococcus, and Ralstonia. The species Propionibacterium acnes and Corynebacterium tuberculostearicum were significantly more abundant in the adult group. Although there were differences in the prevalence and relative abundance of some bacteria observed from adult and child groups, no specific genus or species was detected only in children or adults.