The effects of oral commensal streptococci (Streptococcus sanguinis, Streptococcus gordonii, Streptococcus mitis, and Streptococcus salivarius) on Biofilm formation by cariogenic mutans streptococci (Streptococcus mutans and Streptococcus sobrinus) were investigated. Cell suspensions were cultured on 96-well microtiter plates coated with or without salivary components (SC), and in flow cell systems coated with SC in tryptic soy broth including 0.25% sucrose without dextrose (TSB) . The resultant biofilm formations were stained using safranin or a LIVE/DEAD® BacLightTM Viability Kit, and examined with absorbance at 492 nm or using confocal laser scanning microscopy. Mutans streptococci and S. sanguinis biofilms were formed significantly on the polystyrene surfaces in TSB. Further, in combination cultures, S. sanguinis formed a sufficient biofilm when cultured with S. mutans. However, when S. sanguinis was cultured with S. sobrinus, biofilm formation was slightly inhibited. S. gordonii also inhibited biofilm formation in the culture with S. sobrinus, but not when cultured with S. mutans. S. mitis and S. salivarius collapsed the biofllm morphology and inhibited volume development in some conditions when cultured with S. mutans or S. sobrinus. Biofilm formation by mutans streptococci was challenged and collapsed on the whole by culturing with each of the other oral streptococci. These results indicate that co-culturing of multiple species of mutans streptococci and other oral streptococci has physical effects related to previous attachment and colonization on the surface, as well as biological effects to regulate biofilm formation.