The aim of the present study was to determine the bactericidal effect of a holmium:yttrium-aluminum-garnet (Ho:YAG) laser on root canals in vitro. The efficiency of different laser settings was compared. The in vitro examinations revealed extensive bacterial reduction in extracted, endodontically prepared teeth that had been incubated with Streptococcus faecalis with the most favorable results at a setting of 5 Hz and 2 W. In average, 99.98% of the bacteria injected in the root canal could be eliminated. Considering this bactericidal effect, the application of the Ho:YAG laser in root canal treatment appears to be very efficient.