Bacteria like E. faecalis can produce intra- and extra-radicular biofilms. Theoretically, the adjustable penetration ability of lasers enables better access to root canal system. Therefore the aim of the present study was to compare the ability of photoactivated laser and 2.5% NaOCl irrigation solution to eliminate E. faecalis from the root canals by real-time PCR technique. Sixty extracted human upper central incisors were selected and sterilized in an autoclave. The root canals were infected with E. faecalis (PTCC 1237, Persian Type Culture Collection, Iran) and then incubated for 24 hours. The samples were randomly divided into 3 groups. No intervention was made in the control group (group 1). In group 2, laser therapy was performed with a power of 100 mW by diode laser for 120 seconds. In group 3, the canals were irrigated with 5 mL of 2.5% NaOCl; then all the samples were sonicated in 15 mL of normal saline in test tubes in order to isolate the bacteria. DNA extraction was performed followed by real-time PCR technique for all the samples. Inhibition of bacterial growth in all the experimental samples was significantly more than that in the control group. There was a significant difference between photodynamic therapy and 2.5% NaOCl. The effect of NaOCl in all the samples was better than photodynamic therapy. The results of the mean CT (cyclic threshold) were 40, 30.2 and 15.35 for 2.5% NaOCl, photodynamic therapy and control group, respectively. Based on the results of this experimental study, 2.5% NaOCl eliminated E. faecalis from infected root canals more effectively compared to photodynamic therapy.