The adenoids, primary sites of microbial colonization in the upper airways, can influence the development of various conditions, including otitis media with effusion (OME). Alterations in the adenoid microbiota have been implicated in the pathogenesis of such conditions. This study aims to utilize 16S rRNA genetic sequencing to identify and compare the bacterial communities on the adenoid surfaces of children with OME and children with healthy middle ears. Additionally, we seek to assess the differences in bacterial diversity between these two groups. We collected adenoid surface swabs from forty children, divided into two groups: twenty samples from children with healthy middle ears and twenty samples from children with OME. The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. Alpha and beta diversity indices were calculated, and statistical analyses were performed to identify significant differences in bacterial composition. Alpha diversity analysis, using Pielou's index, revealed significantly greater evenness in the bacterial communities on the adenoid surfaces of the healthy ear group compared with the OME group. Beta diversity analysis indicated greater variability in the microbial composition of the OME group. The most common bacterial genera in both groups were Haemophilus, Fusobacterium, Streptococcus, Moraxella, and Peptostreptococcus. The healthy ear group was primarily dominated by Haemophilus and Streptococcus, whereas the OME group showed higher abundance of Fusobacterium and Peptostreptococcus. Additionally, the OME group exhibited statistically significant higher levels of Alloprevotella, Peptostreptococcus, Porphyromonas, Johnsonella, Parvimonas, and Bordetella compared with the healthy ear group. Our study identified significant differences in the bacterial composition and diversity on the adenoid surfaces of children with healthy middle ears and those with OME. The OME group exhibited greater microbial variability and higher abundances of specific bacterial genera. These findings suggest that the adenoid surface microbiota may play a role in the pathogenesis of OME. Further research with larger sample sizes and control groups is needed to validate these results and explore potential clinical applications.