Statement of problem Proper sterilization or disinfection of removable prostheses and surgical guides has been problematic in dental practice because of the absence of simple and low-cost techniques that do not cause damage to acrylic resins. Purpose The purpose of this study was to study the effect of photodynamic therapy against Streptococcus mutans , Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans biofilms formed on acrylic resin specimens. Material and methods The specimens were sterilized in ethylene oxide gas and submitted to in vitro biofilm growth. The photodynamic therapy consisted of the application of 0.05% methylene blue (P+) conjugated to irradiation with a light-emitting-diode of 630 nm and 150 mW (L+). The specimens were randomly divided into groups (n=5): negative control (P-L-); stained and irradiated at 10 J/cm2 (P+L+ 10); stained and irradiated at 30 J/cm2 (P+L+ 30); stained and not irradiated (P+L-); not stained and irradiated at 10 J/cm2 (P-L+ 10); not stained and irradiated at 30 J/cm2 (P-L+ 30); and gold standard (GS), sterilized. Afterward, the specimens were submitted to contact with culture medium agar for 10 minutes in petri plates, which were incubated for 48 hours at 37°C. The number of colony-forming units was obtained, and the data were expressed according to scores (1=0; 2=1-10; 3=11-100; 4=101-1000) and analyzed by the Friedman and Dunn tests (α=.05). Results Streptococcus mutans was sensitized by (P+L-); P aeruginosa and C albicans were also sensitized by the dye but showed a slight microbial reduction with (P+L+ 30), as did S aureus ( P >.05) ; E coli presented an initial score of 3 and achieved a bacterial reduction to score 2 with (P+L+ 30) ( P =.039). Conclusions Photodynamic therapy was effective in reducing E coli counts on biofilms formed on acrylic resin specimens. The inhibition of microorganism growth tended to be directly proportional to the amount of energy provided by the light-emitting diode.