© 2021 Wiley Periodicals LLCTo investigate the presence of respiratory viruses in the middle ear cavity of the individuals with a healthy middle ear and the children with otitis media with effusion (OME). A total of 72 middle ear samples were collected from 25 children with OME (Group 1) and 47 individuals with no middle ear disease (Group 2). Multiplex real-time polymerase chain reaction was used to investigate the presence of 20 different respiratory viruses. Virus results were compared with bacteriomes of the same populations. At least one respiratory virus was detected in 56% of the patients in Group 1 and 12.8% of the individuals in Group 2. The viral co-infection rate for Group 1 and 2 was 8% and 2.1%, respectively. In Group 1, adenovirus was the most frequently detected virus with a rate of 24%, either alone (16%) or concurrent with other viruses (8%), followed by influenza B (12%), rhinovirus, and bocavirus (8%) each. Parainfluenza 4, coronavirus OC43, and RSV A/B were detected in 4% of the sample each. In Group 2, rhinovirus was detected in two samples (4.3%) followed by adenovirus, coronavirus OC43, coronavirus E299, and coronavirus NL63 with a rate of 2.1% each. The detection rate of respiratory viruses was significantly higher in children aged 6 to 11 years. There was no positive association between virus and bacteria found in the middle ear cavity. The current study has provided comprehensive data indicating the presence of diverse respiratory viruses in the healthy middle ear cavity. Our results also suggest that respiratory viruses might have a contribution to OME pathogenesis.