- Background Co-infections by Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) are frequently implicated in complex otitis media. Whereas upper respiratory-tract carriage precedes disease for both pathogens, interactions between species in co-colonized hosts are poorly understood. We compared colonization densities and the diversity and fitness of pneumococcal serotypes in single-species and mixed-species colonization. Methods We analyzed nasopharyngeal pneumococcal carriage and nasopharyngeal and oropharyngeal NTHi carriage in 13,541 samples collected over 6,909 study visits by 769 children 2-30 months old in a PCV7 dosing trial. We measured density associations between the species and compared pneumococcal serotype diversity during and absent NTHi colonization. We used logistic regression to quantify associations between NTHi colonization and previously-published pneumococcal serotype factors related to fitness. Results Densities of the two species are positively associated when they co-occur in the nasopharynx. NTHi colonization is associated with reduced pneumococcal serotype diversity among children 2-18 months old, and is more prevalent among children carrying pneumococcal serotypes with greater capsular thickness, neutrophil resistance, and metabolic efficiency. Conclusions Pneumococcal-NTHi co-colonization is associated with elevated density of both species, and with reduced diversity and increased fitness of pneumococcal serotypes. NTHi colonization may create a selective environment favoring pneumococci with immune-evasive phenotypes.