OBJECTIVES To evaluate the presence of viruses and bacteria in middle ear and adenoids of patients with and without otitis media with effusion (OME). METHODS Adenoid samples and middle ear washes (MEW) were obtained from children with OME associated with adenoid hypertrophy undergoing adenoidectomy and tympanostomy, and compared to those obtained from patients undergoing cochlear implant surgery, as a control group. Specific DNA or RNA of 9 respiratory viruses (rhinovirus, influenza virus, picornavirus, syncytial respiratory virus, metapneumovirus, coronavirus, enterovirus, adenovirus and bocavirus) and 5 bacteria (S. pneumoniae, H. influenzae, M. catarrhalis, P. aeruginosa and S. aureus) were extracted and quantified by real-time PCR. RESULTS 37 OME and 14 cochlear implant children were included in the study. At the adenoid, virus and bacteria were similarly detected in both OME and control patients. At the middle ear washes, however, a higher prevalence of bacteria was observed in patients with OME (p = 0.01). S. pneumoniae (p = 0.01) and M. catarrhalis (p = 0.022) were the bacteria responsible for this difference. Although total virus detection was not statistically different from controls at the middle ear washes (p = 0.065), adenovirus was detected in higher proportions in adenoid samples of OME patients than controls (p = 0.019). CONCLUSIONS Despite both OME and control patients presented similar rates of viruses and bacteria at the adenoid, children with OME presented higher prevalence of S. pneumonia, M. catarrhalis in middle ear and adenovirus in adenoids when compared to controls. These findings could suggest that these pathogens could contribute to the fluid persistence in the middle ear.