This in vitro study was performed to evaluate the effect of a diode laser and common disinfectants used in combination on mono-infected dental canals. One hundred and six single-rooted human premolars were prepared and contaminated with Enterococcus faecalis. After two weeks of incubation, samples were divided into two experimental groups (n = 48) and two control groups (n = 5). In the first group, the teeth were rinsed for 5 min with either sterile saline, 2.5% NaOCl, or MTAD, or for 1 min with 2% chlorhexidine gluconate (CHX). In the other group, samples were additionally irradiated with a 810-nm diode laser at 2 W output for 5 × 5 s. Intracanal bacterial sampling was done, and the samples were plated to determine the CFU count. In the first group, 2.5% NaOCl was as effective as 2% CHX and significantly more effective than MTAD (P < 0.008). In the second group, either MTAD, 2% CHX or 2.5% NaOCl in combination with laser treatment had a similar effect. Absence of growth was seen only for MTAD plus laser treatment. Complete elimination of E. faecalis was seen only for the combination of MTAD with diode laser irradiation. Combination therapy with MTAD irrigation and diode laser irradiation, within the parameters used in this study, can be recommended as an effective treatment option for complete elimination of E. faecalis from the root canal system. (J Oral Sci 53, 355-360, 2011)