Objectives: Regulatory T lymphocytes (Treg) have been linked to survival of commensal bacteria at mucosal sites, but their presence and role in chronic otitis media (COM) and their response to otopathogens has not been evaluated previously. We investigated the association between Treg lymphocytes and otopathogens in COM prone and non-COM prone children. Methods: Forty children, 2-7 years of age, scheduled for adenoidectomy were enrolled into COM (n = 20) or non-COM (n = 20) groups. Adenoid biopsy and nasopharyngeal aspirate bacteriology were assessed by conventional culture techniques. Peripheral blood and adenoid lymphocytes were stained with viability stain, monoclonal anti-CD19, anti-CD3, anti-CD4, anti-CD8, anti-CD25 and anti-CD127. Cells were stained intracellularly with monoclonal anti-FoxP3 and then quantified by flow cytometry. Results: Children with nasopharyngeal otopathogen-positive culture had significantly more circulating CD3+CD4+FoxP3+CD25hi+CD127lo+ lymphocytes (M = 4.4%) compared to culture-negative children (M = 3.1%, p = 0.005). Circulating CD19+ lymphocytes were significantly increased in children with positive Moraxella catarrhalis nasopharyngeal culture (M = 12.4%) compared to culture-negative children (M = 8.6%, p = 0.006). Adenoid-derived lymphocytes were not significantly different in children with any positive nasopharyngeal culture compared to negative culture. Lymphocyte subsets were not significantly different between COM and non-COM prone children. Conclusion: Clinically-detectable otopathogen nasopharyngeal culture is positively associated with Treg lymphocytes, potentially inducing suppressive effector responses to promote colonization and infection chronicity. This finding supports further investigation of Treg lymphocyte activity and influence on upper airway colonization of young children.