BACKGROUND: Bacteria and respiratory viruses are implicated in the pathogenesis of acute otitis media (AOM); however, data from low-middle income countries are sparse. We investigated the etiology of AOM in HIV-infected (HIV+), HIV-uninfected (HIV-) and HIV-exposed clinically asymptomatic for HIV-infection (HEU) South African children. METHODS: Children >/=3 months to <5 years of age with AOM were enrolled between May 2009 and April 2010 (NCT01031082). Middle ear fluid samples were cultured for bacteria; antibacterial susceptibility was done and serotyping undertaken for Streptococcus pneumoniae and Haemophilus influenzae. Nasopharyngeal aspirates were analyzed for respiratory viruses using immunofluorescence assay and polymerase chain reaction. RESULTS: Of 260 AOM episodes (HIV+:15; HIV-:182; HEU:63), bacteria were found in 54.6%, including Haemophilus influenzae (30.8%), 98.8% of which were nontypeable, and Streptococcus pneumoniae (20.4%), Staphylococcus aureus (15.8%), Moraxella catarrhalis (5.0%) and Streptococcus pyogenes (1.5%). Nonsusceptibility of Streptococcus pneumoniae to penicillin was 64.2%. Respiratory viruses were detected in 74.2% of cases. Human rhinovirus was most frequently detected (37.7%), followed by adenovirus (14.2%) and human bocavirus (11.5%) overall and irrespective of HIV status. Respiratory viruses were identified concurrently with S. pneumoniae, H. influenzae, M. catarrhalis (76.9-78.8%) and Staphylococcus aureus (63.4%) cultured from middle ear fluid, as well as in 72.0% of episodes negative for any bacteria. CONCLUSION: The study suggests that respiratory viruses and pathogenic bacteria play an important role in the development of AOM in children. A similar spectrum of pathogens was observed independently of HIV status. Vaccines targeting both nontypeable Haemophilus influenzae and S. pneumoniae may have a broad impact on AOM in South Africa.