Streptococcus pneumoniae is an important human bacterial pathogen, causing such infections as pneumonia, meningitis, septicemia, and otitis media. Current capsular polysaccharide-based conjugate vaccines protect against a fraction of the over 90 serotypes known, whereas vaccines based on conserved pneumococcal proteins are considered promising broad-range alternatives. The pneumococcal genome encodes two conserved proteins of an as yet unknown function, SP1298 and SP2205, classified as DHH (Asp-His-His) subfamily 1 proteins. Here we examined their contribution to pneumococcal pathogenesis using single and double knockout mutants in three different strains: D39, TIGR4, and BHN100. Mutants lacking both SP1298 and SP2205 were severely impaired in adherence to human epithelial Detroit 562 cells. Importantly, the attenuated phenotypes were restored upon genetic complementation of the deleted genes. Single and mixed mouse models of colonization, otitis media, pneumonia, and bacteremia showed that bacterial loads in the nasopharynx, middle ears, lungs, and blood of mice infected with the mutants were significantly reduced from those of wild-type-infected mice, with an apparent additive effect upon deletion of both genes. Minor strain-specific phenotypes were observed, i.e., deletion of SP1298 affected host-cell adherence in BHN100 only, and deletion of SP2205 significantly attenuated virulence in lungs and blood in D39 and BHN100 but not TIGR4. Finally, subcutaneous vaccination with a combination of both DHH subfamily 1 proteins conferred protection to nasopharynx, lungs, and blood of mice infected with TIGR4. We conclude that SP1298 and SP2205 play a significant role at several stages of pneumococcal infection, and importantly, these proteins are potential candidates for a multicomponent protein vaccine.