ObjectivesStaphylococcus aureusandStreptococcus pneumoniaeare common colonizers of the human nasopharynx. In this study, we describeS. aureusnasopharyngeal carriage and evaluate its association withS. pneumoniaecarriage post-10-valent pneumococcal conjugate vaccine (PCV10) introduction in Pakistan.MethodsA serial cross-sectional study was undertaken from 2014 to 2018, children <2 years were randomly selected, and nasopharyngeal swabs were collected using standard WHO guidelines.S. aureusandS. pneumoniaeisolates were identified using standard methods and tested for antimicrobial susceptibility by the standard Kirby-Bauer disk-diffusion method as per Clinical & Laboratory Standards Institute (CLSI) recommendations. Regression analysis was used to determine predictors associated withS. aureuscarriage.ResultsWe enrolled 3140 children.S. aureuscarriage prevalence was 5.6% (176/3140), and 50.1% (81/176) of the isolates were methicillin-resistantS. aureus(MRSA).S. aureuscarriage was higher in the absence of pneumococcus compared to isolates in which pneumococcus was present (7.5% vs 5.0%).S. aureuscarriage was negatively associated with pneumococcal carriage, being in 3rd and 4th year of enrollment, and vaccination with two and three PCV10 doses, in addition, fast breathing, ≥2 outpatients visits, and rainy season were positively associated. The following resistance rates were observed: 98.9% for penicillin, 74.4% for fusidic acid, and 23.3% for gentamicin, 10.2% for erythromycin, and 8.5% for cotrimoxazole. All isolates were susceptible to amikacin.ConclusionsOverallS. aureuscarriage prevalence was low, PCV10 vaccine was protective against the carriage. The proportion of MRSA carriage and antimicrobial resistance was high in this community warranting continuous monitoring for invasive infections.