To study the extent of surface adenoid biofilm and to evaluate its role in the pathogenesis of chronic otitis media with effusion (COME) in children. The study was carried out on 100 children between 3 and 14 years of age, who were divided into two groups. The first group (50 children) had otitis media with effusion associated with adenoid hypertrophy, whereas the second group (50 children) had adenoid hypertrophy without middle ear effusion. Adenoidectomy with ventilation tube insertion was done for group 1 cases, whereas, only Adenoidectomy was done for group 2 cases. Microbiological study, Scanning electron microscope and multiplex- PCR were done for suspected adenoid biofilms and specimens from middle ear effusion. Adenoids removed from children with COME had higher grade biofilm formation (74 %) than the second group (42 %). No correlation was found between adenoid size and biofilm formation. Culture of adenoid tissue in group 1 patients was positive in 52 % of cases compared to 96 % by PCR, while in group 2 culture of adenoid tissue was positive in 38 % compared to 48 % by PCR. Culture of middle ear fluid was positive in 32 % of cases only compared to 80 % by PCR. A positive correlation was found between results of bacterial biofilm visualized by SEM and bacteria detected and identified by PCR technique. On the other hand, no correlation was found between results of bacterial biofilm visualized by SEM and bacteria detected by culture. The size of the adenoid is not the main determinant factor in OME pathogenesis but the degree of bacterial colonization is much more important. Adenoids in COME may act as a reservoir of chronic infection rather than causing mechanical Eustachian obstruction. Higher grade biofilm formation was found in cases with middle ear effusion than those with adenoid hypertrophy only. These findings support the hypothesis that there would be an association between adenoidal biofilm formation and COME. This study focused on the value of PCR in detecting pathogens in the adenoid and middle ear specimens although the bacterial culture would be negative.