The capability of Nd:YAG laser in sterilizing root canals and the alterations of dentinal walls induced by laser treatment were investigated. Thirty root canals were infected by P. aeruginosa ATCC 27853 and thirty canals by A. naeslundii CH-12. Within each infection, 4 groups were selected on the basis of the treatment. Among them, 2 test groups (TGs) were treated by Nd:YAG laser at 15 Hz for 15 s, using 2 different settings: 1 Watt/70 Joule and 1.5 Watt/100 Joule, respectively (n = 10 each). The other 2 groups, used as controls (CGs), were: untreated (positive control, n = 5) and sterilized by 5.25% NaClO group (negative control, n = 5). Observations under scanning electron microscope (SEM) and quantitative bacterial counts were performed. These analyses were performed once per group after infections and treatments. Laser treatments significantly reduced the number of both bacteria. SEM investigation showed melting and crystallization of canal dentin over 1.5 W/100 J. Laser irradiation has a bactericidal effect but it does not completely sterilize the root canal as NaClO 5.25% solution does if the goal of treatment is also to avoid alterations of dentinal walls.