The antimicrobial effect of photodynamic therapy (PDT) using conventional lasers is well established. Recently, there has been an increasing interest in replace lasers by cheaply lights such as Light Emission Diodes (LED). Objectives: The aims of this study were firstly to standardize a Streptococcus mutans, Streptococcus sobrinus and Streptococcus sanguinis biofilm model; and then, evaluate the effect of a red LED associated with toluidine blue O (TBO) on the viability of these biofilms. Methods: The biofilms of related bacteria were formed at a solid-liquid interface using enamel slabs as substrate and having a periodic replacement of nutrient media during 3, 5 or 7 days. Thus, the microbiological counting and water-insoluble polysaccharide (WIP) concentrations were analyzed in the biofilms; while mineral loss (DZ) analysis was performed on enamel slabs. In addition, the antimicrobial potential of 100 mg.ml-1 TBO associated with 85.7 J.cm-2 LED light (spectrum of emission ranging from 620 to 660 nm and a 638.8 nm predominant wavelength.) on viability of 5-day biofilms were evaluated. Controls without exposure to LED/TBO and exposed only to LED or TBO were also performed. Results: The results showed that the analyzed variables increased in accordance with the age of biofilms as follows: the number of microorganisms (1.8x105 to 5.4x107 CFU.mg-1), DZ (2566.9 to 5686.6 vol%min.mm) and WIP concentration (194.6 to 459.9 mg.g-1). Significant reductions on viability of microorganisms of 94.96%, 95.08% and 99.97% were found respectively by S. mutans S. sobrinus and S. sanguinis biofilms, after photosensitization. Conclusions: The biofilm model was shown to be suitable for studying changes in bacterial numbers and enamel mineralization and for demonstrating the potential value of photosensitization in the control of in vitro biofilms. (Supported by FAPES 02/00393-8).