This study analyzed the necessity of use of an optical fiber/diffusor when performing antimicrobial photodynamic therapy (PDT) associated with endodontic therapy. Fifty freshly extracted human single-rooted teeth were used. Conventional endodontic treatment was performed using a sequence of ProTaper (Dentsply Maillefer Instruments), the teeth were sterilized, and the canals were contaminated with Enterococcus faecalis 3 days' biofilm. The samples were divided into five groups: group 1--ten roots irradiated with a laser tip (area of 0.04 cm(2)), group 2--ten roots irradiated with a smaller laser tip (area of 0.028 cm(2)), and group 3--ten teeth with the crown, irradiate with the laser tip with 0.04 cm(2) of area. The forth group (G4) followed the same methodology as group 3, but the irradiation was performed with smaller tip (area of 0.028 cm(2)) and G5 ten teeth with crown were irradiated using a 200-mm-diameter fiber/diffusor coupled to diode laser. Microbiological samples were taken after accessing the canal, after endodontic therapy, and after PDT. Groups 1 and 2 showed a reduction of two logs (99%), groups 3 and 4 of one log (85% and 97%, respectively), and group 5 of four logs (99.99%). Results suggest that the use of PDT added to endodontic treatment in roots canals infected with E. faecalis with the optical fiber/diffusor is better than when the laser light is used directed at the access of cavity.