The in vitro reduction of the bacterial population in the root canal by the mechanical action of instrumentation and irrigation was evaluated. Root canals inoculated with a Enterococcus faecalis suspension were instrumented using hand Nitiflex files, Greater Taper (GT) files, and Profile 0.06 taper Series 29 rotary instruments. Irrigation was performed using sterile saline solution. Root canals were sampled before and after instrumentation. In the group of the Nitiflex files, samples were also taken after each file size. After serial dilution, samples were plated onto Mitis-Salivarius agar, and the colony forming units grown were counted. All techniques and instruments tested were able to reduce significantly the number of bacterial cells in the root canal. Instrumentation to a Nitiflex #30 was significantly more effective than GT files. There were no significant differences when comparing the effects of the Profile instrument #5 with either the GT files or the Nitiflex #30. Enlargement to a Nitiflex #40 was significantly more effective in eliminating bacteria when compared with the other techniques and instruments tested (p < 0.05). The results of this study showed that the instrumentation and irrigation can mechanically remove more than 90% of bacterial cells from the root canal.