The purpose of this study was to investigate the antimicrobial properties of Er:YAG-laser radiation in dental root canals. The root canals of 90 freshly extracted anterior teeth were enlarged mechanically, sterilized, and randomly divided into subgroups of 10 samples. The root canals were inoculated with Escherichia coli or Staphylococcus aureus for 2 h. The laser treatment groups were exposed for either 15 or 60 s to Er:YAG-laser radiation (pulse energy: 50 mJ; 15 pps). Additionally, for each bacterial strain, one sample group was rinsed with a NaOCl solution (1.25%), and one was left untreated as control. After irradiation or irrigation, the number of bacteria was evaluated using the surface spread plate technique. In the case of S. aureus, the primary bacterial load (control group) of the root canals was reduced to 0.15% after 15 s and 0.06% after 60 s of laser treatment. In the E. coli group, the number of bacteria was diminished to 0.13%, with the shorter radiation time and to 0.034% after 60 s of radiation. Irrigating the root canals with NaOCl, a reduction of the number of bacteria to 0.033% for S. aureus and to 0.020% for E. coli could be obtained. As the results confirm, Er:YAG-laser radiation exerts very effective antimicrobial properties in dental root canals, depending on the time of radiation.