The clinical significance of pneumococcal biofilm formation is largely unknown. To clarify this, we tested whether the ability of pneumococcal clinical isolates to form biofilm in vitro accounts for the diverse clinical outcomes. Clinical pneumococcal isolates were cultured from the nasopharynx (n=106), middle ear effusion (n=43) and blood (n=55) of 204 children altogether. Biofilm formation, assessed by measuring optical density (OD) values in microtitre plates after crystal violet staining, did not differ between the bacteria from different sources (p=0.18), the mean OD values of the isolates being 0.119 [95% confidence interval (CI) 0.100-0.138] in the nasopharynx samples, 0.094 (95% CI 0.069-0.119) in the acute otitis media cases, 0.109 (95% CI 0.077-0.141) in the secretory otitis media cases, 0.122 (95% CI 0.084-0.160) in those with sepsis and 0.175 (95% CI 0.071-0.280) in those with other invasive infections. Serotypes 33 and 14 were the most efficient in forming biofilms, whereas serotypes 3 and 38 were poor biofilm producers. We conclude that the clinical presentation of pneumococcal disease did not differ in relation to biofilm formation in vitro, even though there was marked variation between the clinical isolates and serotypes.