Streptococcus mutans is one of the principal pathogens for the development of dental caries. Oral biofilms formed by S. mutans are constructed of insoluble glucan formation induced by the principal enzymes, GTF-I and GTF-SI, in sucrose-containing conditions. However, as another means of biofilm formation, extracellular DNA (eDNA) and membrane vesicles (MVs) are also contributors. To explore the roles of eDNA and MVs for biofilm formation, short and whole size pure DNAs, two types of sub-purified DNAs and MVs were extracted from S. mutans by beads destruction, treatment of proteinase K, and ultracentrifugation of culture supernatant, and applied into the biofilm formation assay using the S. mutans UA159 gtfBC mutant, which lost GTF-I and GTF-SI, on a human saliva-coated 96 well microtiter plate in sucrose-containing conditions. Sub-purified DNAs after cell lysis by beads destruction for total 90 and 180 s showed a complex form of short-size DNA with various proteins and MVs associated with GTF-I and GTF-SI, and induced significantly higher biofilm formation of the S. mutans UA159.gtfBC mutant than no sample (p < 0.05). Short-size pure DNA without proteins induced biofilm formation but whole-size pure DNA did not. Moreover, the complex form of MV associated with GTFs and short-size DNA showed significantly higher biofilm formation of initial colonizers on the human tooth surface such as Streptococcus mitis than no sample (p < 0.05). The short-size DNAs associated with MVs and GTFs are important contributors to the biofilm formation and may be one of additional targets for the prevention of oral biofilm-associated diseases.