Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus are commonly carried in the nasopharynx of young children, and have been speculated to interact with each other. Although earlier studies used cultures alone to assess these interactions, the addition of real-time quantitative polymerase chain reaction (qPCR) provides further insight into these interactions. We compared results of culture and qPCR for the detection of these 3 bacteria in 446 nasopharynx samples collected from 360 healthy young children in a prospective cohort study in the Peruvian Andes. Patterns of concurrent bacterial colonization were studied using repeated measures logistic regression models with generalized estimating equations. Spearman correlation coefficients were used to assess correlations among bacterial densities. At a bacterial density <10 colony forming units/mL measured by qPCR, culture detected significantly less carriers (P < 0.0001) for all 3 pathogens, than at a bacterial density >10 colony forming units/mL. In addition, there was a positive association between S. pneumoniae and H. influenzae colonization measured by both culture (odds ratio [OR] 3.11-3.17, P < 0.001) and qPCR (OR 1.95-1.97, P < 0.01). The densities of S. pneumoniae and H. influenzae, measured by qPCR, were positively correlated (correlation coefficient 0.32, P < 0.001). A negative association was found between the presence of S. pneumoniae and Staphylococcus aureus in carriage with both culture (OR 0.45, P = 0.024) and qPCR (OR 0.61, P < 0.05). The impact of density on detection by culture and the observed density-related interactions support use of qPCR in additional studies to examine vaccine effects on diverse bacterial species. © 2012 Lippincott Williams & Wilkins.