The present study was performed to elucidate the clinical outcome, and etiology of acute otitis media (AOM) in children based on virologic and bacteriologic tests. The study group consisted of 120 children aged 6 to 144 months with AOM. Middle ear fluid (MEF) was tested for viral pathogens by reverse transcriptase polymerase chain reaction (RT-PCR) and for bacteria by gram-staining and culture. Clinical response was assessed on day 2 to 4, 11 to 13, 26 to 28. Respiratory viruses were isolated in 39 patients (32.5%). Respiratory syncytial virus (RSV) (46.5%) was the most common virus identified in MEF samples, followed by human rhinovirus (HRV) (25.6%), human coronavirus (HCV) (11.6%), influenza (IV) type A (9.3%), adenovirus type sub type A (AV) (4%), and parainfluenza (PIV) type -3 (2%) by RT-PCR. In total 69 bacterial species were isolated from 65 (54.8%) of 120 patients. Streptococcus pneumoniae (S. pneumoniae) was the most frequently isolated bacteria. Viral RNA was detected in 31 (56.3%) of 55 bacteria-negative specimens and in 8 (12.3%) of 65 bacteria-positive MEF samples. No significant differences were found between children representing viral infection alone, combined viral and bacterial infection, bacterial infection alone, and neither viral nor bacterial infection, regarding clinical cure, relapse and reinfection rates. A significantly higher rate of secretory otitis media (SOM) was observed in alone or combined RSV infection with S. pneumonia or Haemophilus influenzae (H. influenzae) than in other viruses infection. Conclusion. This study provides information about etiologic agents and diagnosis of AOM in Turkish children. The findings highlight the importance of common respiratory viruses and bacterial pathogens, particularly RSV, HRV, S. pneumoniae and H. influenzae, in predisposing to and causing AOM in children.