- Background: Complex (ie, recurrent, nonresponsive, or chronic) otitis media (OM) is frequent and is often caused by a mixed-pathogen infection with biofilm formation. We conducted this study to characterize children with OM due to mixed Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) infections (M-OM) and those with OM due to single, S. pneumoniae-only infections (S-OM) and to examine whether pneumococcal serotypes associated with M-OM differed from those associated with S-OM. Methods: In a 10-year prospective study in southern Israel, the clinical and demographic variables and pneumococcal serotypes associated with M-OM were compared to those associated with S-OM in children <3 years old. Results: M-OM episodes were significantly more likely to be found in Bedouin children (for whom living conditions are crowded and colonization occurs during early life) and in older children with bilateral OM, recurrent OM, previous tympanocentesis, and lower body temperature, as well as during the winter, suggesting an association with recurrence/chronicity. M-OM was associated with pneumococcal serotypes most commonly carried by healthy children, whereas S-OM was associated with serotypes previously shown to have a higher disease potential. Conclusions: S-OM and M-OM differ clinically and epidemiologically, with overlapping characteristics. Our findings are in agreement with clinical and experimental reports associating respiratory tract biofilms and mixed infections with pneumococcal serotypes of lower virulence and higher capacity to colonize the nasopharynx in healthy individuals.