Background & aim: Adenoid hypertrophy is one of the childhood complications in which bacteria are involved in its etiology. The aim of the present study was to determine the relative abundance of biofilms and genes associated with biofilm formation in Staphylococcus aureus isolates and also to determine the genotypic patterns of S. aureus isolates from adenoids and tonsils of children with adenoid hypertrophy by Spa typing method. Methods: The present cross-sectional study was conducted on 86 pair isolates of S. aureus collected from adenoid and tonsil of children with adenoid hypertrophy. Phenotypic method used for assessing of biofilm production in S. aureus isolates and the presence of icaA, icaD, fnbA and clfA genes evaluated using polymerase chain reaction method. The spa typing method was apllied for typing of isolated. Data were analyzed using descriptive statistics method and SPSS software. Results: S. aureus isolated from adenoids and tonsils were positive in 49 (57%) and 40 (46.5%) biofilms, respectively. In 47 cases, pairs of isolates from the adenoids and tonsils were similar in terms of biofilm production. In the present study, 4 types of spa types were identified in S. aureus including t081 type, the most dominant type in 67.5%, t701 in 11.6%, t2419 in 9.3% and t4870 in 8.2% of adenoid isolates. In total, 79.45% of adenoid and tonsil isolates had similar typing. highest frequency of genes was related to fnbA, which was detected in adenoids in 64% and in tonsils in 41.9% of isolates. Conclusion: Due to the relative frequency of biofilm formation, biofilm is of moderate importance in the establishment of Staphylococcus aureus in the tonsils and adenoids of patients. Moreover, the role of fnbA gene in biofilm formation was higher than other studied genes. High spa similarity Staphylococcus aureus isolates isolated from the adenoids and tonsils indicate the initial colonization of bacteria in the tonsils and then act as a source of infection for the adenoids