Background: The upper respiratory tract harbors diverse communities of commensal, symbiotic, and pathogenic organisms, originating from both the oral and nasopharyngeal microbiota. Among the primary sites of microbial colonization in the upper airways are the adenoids. Alterations in the adenoid microbiota have been implicated in the development of various conditions, including secretory otitis media. Aim: This study aims to employ 16S rRNA genetic sequencing to identify the most common bacteria present on the surface of adenoids in children with otitis media with effusion and compare them with children without pathologies in the tympanic cavity. Additionally, we seek to determine and compare the bacterial diversity in these two study groups. Materials and Methods: A total of nineteen samples from the adenoid surfaces were collected, comprising two groups: thirteen samples from children without middle ear effusion and six samples from children with secretory otitis media. The libraries of the V3–V4 hypervariable region of the bacterial 16S rRNA gene was made and sequenced using MiSeq platform. Results: The most prevalent phyla observed in both groups were Proteobacteria, Firmicutes, and Bacteroidetes. The most common bacterial genera identified in both groups were Haemophilus, Streptococcus, Moraxella, Fusobacterium, and Bordetella, with Fusobacterium and Moraxella being more prevalent in the groups that had no middle ear effusion, while Haemophulus and Streptococcus were more prevalent in the otitis media with effusion group, although not in a statistically significant way. Statistical analysis shows a trend towards bacterial composition and beta diversity being similar between the study groups; however, due to the limited sample size and unevenness between groups, we should approach this data with caution. Conclusion: The lack of prolific difference in bacterial composition between the study groups suggests that the role of the adenoid microbiome in the development of otitis media with effusion may be less significant.