Biofilm formation has been suggested to play an important role during Streptococcus pneumoniae nasopharyngeal colonization and may facilitate progression to pneumonia. To test whether the ability of S. pneumoniae to form biofilms was important for virulence we screened the ability of 30 invasive and 22 non-invasive clinical isolates of serotype 6A and 6B to form early biofilms on polystyrene microtiter plates and infect mice following intranasal and intratracheal challenge. We first determined that no correlation existed between the ability to form early biofilms and whether isolates were collected from healthy carriers or individuals with invasive disease. A disconnect between biofilm forming ability and the capacity to colonize the nasopharynx, cause pneumonia, and enter the bloodstream was also observed in mice. Importantly, S. pneumoniae mutants deficient in the established virulence determinants pneumolysin, CbpA, and hydrogen peroxide formed biofilms normally. Incidentally, we determined that robust biofilm production was dependent on the formation and coalescing of bacterial aggregates on a thin layer of bacteria attached to the plate surface. In summary, these studies suggest that the ability to form early biofilms in vitro does not reflect virulence potential. More complex studies are required to determine if biofilm formation is important for virulence. © 2010 Elsevier Ltd. All rights reserved.