Acute otitis media with effusion (OME) is one of the major causes of antibiotic use, indication for operation and hearing loss in children. In two third of the cases the etiologic agents are bacteria. Nonetheless, increasing numbers of reports have implicated viruses as etiologic agents that may have some effect on prognosis of OME. The aim of this study was to investigate the presence of nucleic acids of respiratory syncytial virus (RSV) type A and B, influenza type A virus, adenovirus, cytomegalovirus (CMV), herpes simplex virus type-1 (HSV-1), and enteroviruses in the middle ear effusion specimens from children with otitis media by TaqMan real-time PCR. As a result, 18 of 30 (60%) OME samples were found positive in terms of viral nucleic acids by real-time PCR. RSV-A was detected in nine samples (30%), CMV in 3 (10%) samples and HSV-1 in 1 (3.3%) sample. In five of the samples two viruses were detected in the same sample (three were positive for adenovirus and RSV-A, and two were positive for CMV and RSV-A). Our data have supported the importance of viruses as etiologic agents of OME. Additionally, it was thought that TaqMan real-time PCR may be used as a reliable and rapid method for the detection of viruses in the middle ear effusion samples.