The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone-like DNA-binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity. Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7-hydoxyl-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) and anti-HLP antibody without fixation, respectively. DNase I treatment (200 U ml(-1)) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml(-1)) purified from Strep. intermedius, other Gram-positive bacteria, Gram-negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild-type, HLP-downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml(-1)) decreased the biofilm mass of all Strep. intermedius strains. These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity. eDNA- and HLP-targeting strategies may be applicable to novel treatments for bacterial biofilm-related infectious diseases. © 2013 The Society for Applied Microbiology.