The current study investigates the bactericidal effect of a pulsed Nd:YAG laser on 40, classically prepared, in vitro root canals under standardized conditions. The output of the device including quartz fiber was measured before and after every treatment to exclude any technical faults. A 300-micron fiber was used for the anterior teeth and a 200-micron fiber for the posterior teeth. The Enterococcus faecalis bacteria, as gram-positive cocci, display a heat tolerance in the upper range of the bacilli that can be found in the endodontology and the concentration also reflects the in vivo conditions. The results are quoted in "log kills" in accordance with the work of Rooney et al. On average, 99.91% of the Enterococcus faecalis bacteria injected into the root canals prepared in vitro could be eliminated (97.12% minimum, 99.9997% maximum). This setup suggests that the bactericidal effect of laser therapy in the framework presented here can also be achieved in vivo in clinical cases.