Breastfeeding elicits significant protection against respiratory tract infections in infancy. Modulation of respiratory microbiota might be part of the natural mechanisms of protection against respiratory diseases induced by breastfeeding. To study the association between breastfeeding and nasopharyngeal microbial communities, including all cultivable and noncultivable bacteria. In this observational study, we analyzed the microbiota of infants that had received exclusive breastfeeding (n = 101) and exclusive formula feeding (n = 101) at age 6 weeks and 6 months by 16S-based GS-FLX-titanium-pyrosequencing. At 6 weeks of age the overall bacterial community composition was significantly different between breastfed and formula-fed children (nonmetric multidimensional scaling, P = 0.001). Breastfed children showed increased presence and abundance of the lactic acid bacterium Dolosigranulum (relative effect size RES, 2.61; P = 0.005) and Corynebacterium (RES, 1.98; P = 0.039) and decreased abundance of Staphylococcus (RES, 0.48; P 0.03) and anaerobic bacteria, such as Prevotella (RES, 0.25; P < 0.001) and Veillonella (RES, 0.33; P < 0.001). Predominance (>50% of the microbial profile) of Corynebacterium and Dolosigranulum was observed in 45 (44.6%) breastfed infants compared with 19 (18.8%) formula-fed infants (relative risk, 2.37; P = 0.006). Dolosigranulum abundance was inversely associated with consecutive symptoms of wheezing and number of mild respiratory tract infections experienced. At 6 months of age associations between breastfeeding and nasopharyngeal microbiota composition had disappeared. Our data suggest a strong association between breastfeeding and microbial community composition in the upper respiratory tract of 6-week-old infants. Observed differences in microbial community profile may contribute to the protective effect of breastfeeding on respiratory infections and wheezing in early infancy. Clinical trial registered with www.clinicaltrials.gov (NCT 00189020).