The aim of this in vitro study was to compare the efficacy of a classical irrigant with that of a laser in disinfecting a contaminated root canal. Thirty canals of extracted single-root teeth were prepared with files to size #20. The teeth were sterilized with Germispad (Spad, France) for 30 min and then inoculated with Streptococcus mitis ATCC 33399. By randomization, the teeth were divided into six groups of five teeth each. In the first group, teeth were neither inoculated nor prepared. This was the sterility control group (1). In the second group (2) teeth were inoculated without any preparation: as positive controls. The third group was inoculated and then hand-instrumented with files to size #30 with 5.25% NaOCl as irrigant. This was the hand instrumentation group. The other groups were prepared with hand instrumentation with files to size #30, using sterile water as an irrigant, and the canal was then lased with different frequencies as follows: group 4, frequency of 5 Hz and power of 260 mJ; group 5, frequency of 10 Hz and power of 310 mJ; and group 6, frequency of 30 Hz and power of 300 mJ. After experimentation, the residual colonies were counted. The results indicated that (i) the treatment with NaOCl and manual instrumentation effectively inhibited the growth of Streptococcus mitis ATCC 33399; and (ii) the antibacterial effect of the Nd:YAP laser depended on the frequency. Only a frequency of 30 Hz of the Nd:YAP laser inhibited the growth of Streptococcus mitis ATCC 333999.