Aim: The present study was conducted to assess the effects of Er: YAG laser irradiation on root canals in vitro. Materials and Methods: 200 extracted human teeth were endodontically processed and subsequently irradiated at different settings using an Er:YAG laser imitating in vivo irradiation procedures. The teeth were then subdivided into three groups and subjected to bacteriological evaluations, scanning electron microscopy, and temperature measurements. Results: The bacteriological evaluation revealed a decisive bactericidal effect of the Er:YAG laser in the root canal. The bactericidal effect was dependent on the applied output power and specific for the different species of bacteria investigated. Scanning electron microscopy showed discrete removal of dentine from the root canal walls. The temperature rise during irradiation was moderate when standardized power settings were used. Conclusion: The investigations indicate that the Er:YAG laser is a suitable tool for the elimination of bacteria in root canals under in vitro conditions.