According to studies based on bacterial cultures of middle ear fluids,Streptococcus pneumoniae,Haemophilus influenzae, andMoraxella catarrhalishave been the most common pathogens in acute otitis media. However, bacterial culture can be affected by reduced viability or suboptimal growth of bacteria. PCR detects bacterial DNA from samples with greater sensitivity than culture. In the present study, we analyzed the middle ear pathogens with both conventional culture and semiquantitative real-time PCR in 90 middle ear fluid samples obtained from children aged 5 to 42 months during acute otitis media episodes. Samples were tested for the presence ofS. pneumoniae,H. influenzae,M. catarrhalis,Alloiococcusotitidis,Staphylococcus aureus, andPseudomonas aeruginosa. One or more bacterial pathogens were detected in 42 (47%) samples with culture and in 69 (77%) samples with PCR. According to PCR analysis,M. catarrhalisresults were positive in 42 (47%) samples,H. influenzaein 30 (33%),S. pneumoniaein 27 (30%),A. otitidisin 6 (6.7%),S. aureusin 5 (5.6%), andP. aeruginosain 1 (1.1%). Multibacterial etiology was seen in 34 (38%) samples, andM. catarrhaliswas detected in most (85%) of those cases. Fifteen signals forM. catarrhaliswere strong, suggesting a highly probable etiological role of the pathogen. In conclusion, even thoughM. catarrhalisis often a part of mixed flora in acute otitis media, a considerable proportion of cases may be primarily attributable to this pathogen.