PURPOSE: Helicobacter pylori, causing chronic systemic infection, may colonize in middle ear milieu and conduce to effusion collection. Many investigations on relationship between pathogenesis of otitis media with effusion (OME) and Helicobacter pylori yielded conflicting results. We investigated Helicobacter pylori presence in effusion and adenoid samples of children having OME and in middle ear and adenoid samples of children with healthy middle ears to elucidate its role on OME pathogenesis. METHODS: This prospective case-control study included 300 patients aged 1-12 years. One-hundred effusion samples collected from 100 children undergoing ventilation tube insertion and adenoidectomy due to chronic OME and adenoid hypertrophy formed study group, and 100 adenoid samples collected from adenoids of these children formed Group-1. One-hundred healthy-looking middle ear irrigation solutions collected from 100 children undergoing cochlear implantation formed Group-2. One-hundred adenoid samples collected from 100 children having no effusion and only undergoing adenoidectomy formed Group-3. After DNA isolation of samples, Helicobacter pylori 16 S rRNA and 23 S rRNA gene for clarithromycin-resistance were investigated by real time-polymerase chain reaction (Rt-PCR). RESULTS: The median age of 300 children was 5, and 179 were boys and 121 were girls. Helicobacter pylori was detected by Rt-PCR in none (%0) of the 400 samples (200 middle ear, 200 adenoid). CONCLUSION: In this largest sample-size study utilizing updated molecular methods to date, negative results indicate that Helicobacter pylori does not play role as an active pathogen in polymicrobiality of OME, and adenoids do not serve as a reservoir for Helicobacter pylori in this process.