Nasopharyngeal colonization represents the initial interaction between Haemophilus influenzae and its human host. Factors that influence bacterial carriage likely affect transmission and incidence of infection. Therefore, we investigated host factors involved in limiting H. influenzae colonization in BALB/c mice, as colonization can be established in this genetic background. Unlike what is observed in the C57BL/6 background, initial colonization of BALB/c mice was mainly limited by adaptive immune components. This effect on colonization did not require either CD4- or CD8-positive T cells. Instead, initial colonization by genetically diverse strains was limited by preexisting natural antibody with a lesser contribution of complement activity and the presence of neutrophils. Natural serum immunoglobulin from mice was able to bind to the bacterial surface and exhibited complement-dependent bactericidal activity against these genetically diverse H. influenzae strains. Moreover, natural immunoglobulin G (IgG) recognizing these strains was detected at the nasopharyngeal mucosal surface. This antibody-mediated effect required exposure to the normal mouse microbial flora, since mice raised under germfree (GF) conditions showed increased levels of H. influenzae colonization that were not limited by adaptive immunity. In addition, serum IgG from GF mice exhibited less surface binding to H. influenzae, suggesting that natural antibody, induced through prior exposure to the microbial flora, mediated the observed reduction in initial colonization. The broad effect of natural IgG against genetically diverse isolates suggests the presence of conserved species-wide protective targets of antibody.