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Objectives Aim of our study was to map the adenotonsillar lymphoid tissues’ microbiome identifying its potential etiopathogenetic role in children affected by chronic tonsillitis or tonsillar hypertrophy with Obstructive Sleep Apnea Syndrome (OSAS). Methods In our study, we examined tonsillar swabs from healthy children and children affected by chronic tonsillitis or by tonsillar hypertrophy with Obstructive Sleep Apnea Syndrome (OSAS). Microbiome's analysis was performed and bacterial 16Sr RNA gene was sequenced according to metagenomic principles. Variability was described according to the biodiversity concept, indicating species found in a certain environment and changes they undergo adapting to different environmental conditions. Results The most significant differences concern variation of microbes in a single sample (alpha diversity) of some phyla in children affected by chronic tonsillitis compared with alpha diversity in healthy children and in children affected by OSAS with tonsillar hyperplasia. Proteobacteria are prevalent in chronic tonsillitis group, Fusobacteria and Spirochete in OSAS and Firmicutes, Actinobacteria, and Bacteroidetes were found in healthy children. Finally, comparison between the groups showed that children with OSAS with tonsillar hypertrophy had a higher presence of the Fusobacterium genus. Conclusion Recurrent upper airway inflammatory and/or infectious processes are polymicrobial; chronicity of such processes appear to be related to variations in microbiome's composition and interaction among various taxonomic units. Knowledge of the microbiomes’ composition together with traditional clinical biomarkers can also determine relationships between oropharyngeal microbiome and systemic pathologies to determine preventive changes in lifestyle, eating habits, environmental exposure and use of probiotics.