BACKGROUND Acute otitis media (AOM) is a main cause for antimicrobial prescription in Latin America. Pathogen diversity in different geographic regions underscores the need for updated knowledge on AOM microbiology. OBJECTIVE To prospectively determine the role of bacteria and viruses in Chilean children with AOM. METHODS Between July, 1998, and June, 1999, children >3 months with a presumptive diagnosis of AOM were referred to the study ear, nose and throat physician. Middle ear fluid and nasopharyngeal aspirates were obtained from children with confirmed AOM and processed for common bacteria, Mycoplasma pneumoniae, Chlamydia pneumoniae and viruses. Antimicrobial susceptibility patterns and serotypes of Streptococcus pneumoniae strains were determined. RESULTS An ear, nose and throat physician confirmed diagnoses for 222 (42%) of 529 children referred with diagnosis of AOM, and 170 children met eligibility criteria for the study. One or more pathogens were detected in 140 of 170 (82%) children. Predominant bacteria were S. pneumoniae (37%), Haemophilus influenzae (24%) and Streptococcus pyogenes (13%). M. catarrhalis was detected in 2 children, C. pneumoniae was found in 1 and M. pneumoniae was not detected. Viruses were detected in 22 children (13%) from nasopharyngeal aspirates, and in 6 of them the same virus was detected in middle ear fluid. Penicillin-resistant (intermediate and high) S. pneumoniae represented 40% of isolates and 10% of H. influenzae were beta-lactamase producers. All 10 penicillin-resistant S. pneumoniae strains were resistant to cefuroxime. Eighteen S. pneumoniae serotypes were detected and 19F was associated with high level penicillin resistance. CONCLUSIONS This study can impact local management of AOM, and it should encourage continuous surveillance of AOM microbiology in Chile and other developing countries.