Purpose: Chronic otitis media with effusion (OME) is the leading cause of hearing loss during childhood. In bacterial etiology of OME, the most frequent pathogens responsible are Haemophilus influenzae followed by Streptococcus pneumoniae and Moraxella catarrhalis . This study aimed at evaluating the accuracy of nasopharyngeal (NP) specimens in the identification of pathogens in the middle ear fluid (MEF) in patients with OME. Materials and Methods: In this cross sectional, case-control study, 95 MEFs and 53 NP secretion specimens were obtained from 53 children. As a control group, 102 NP specimens were taken from children having an operation other than an otological disease. Conventional culture methods and multiplex-PCR method have been used to determine the etiology of OME; NP carriage between cases and control groups were compared using conventional culture methods. Pearson Chi-Square and Fishers Exact tests were used in statistical analysis. Results : Bacteria were isolated by culture in 37.9 of MEF specimens, 14.7 of which belonged to the group H. influenzae , S. pneumoniae and M. catarrhalis. PCR was positive in 30.5 specimens targeting the same pathogens. There was a two-fold increase in carriage rate of S. pneumoniae and H. influenzae in patients than controls for each pathogen. Conclusion: PCR is a more reliable method to detect middle ear pathogens in MEF in comparison with the conventional culture methods. The NP colonization wasnt found to be an indicator of the pathogen in MEF although middle ear pathogens colonize more in nasopharynx of diseased children.