Objective: To compare the disinfecting efficacy of the sodium hypochlorite irrigation by root canal preparation with stainless steel hand files, taper 0.02 and nickel-titanium Mtwo files with taper 0.04-0.06. Study design: 40 extracted human teeth were sterilized, and then inoculated with Enterococcus faecalis (ATCC 29212). After 6 day incubation time the root canals were prepared by hand with K-files (n=20) and by engine-driven Mtwo files (VDW, Munich, Germany) (n=20). Irrigation was carried out with 2.5% NaOCl in both cases. Samples were taken and determined in colony forming units (CFU) from the root canals before and after the preparation with instruments #25 and #35. Results: Significant reduction in bacterial count was determined after filing at both groups. The number of bacteria kept on decreasing with the extension of apical preparation diameter. There was no significant difference between the preparation sizes in the bacterial counts after hand or engine-driven instrumentation at the same apical size. Statistical analysis was carried out with Mann-Whitney test, paired t-test and independent sample t-test. Conclusions: Significant reduction in CFU was achieved after the root canal preparation completed with 2.5% NaOCl irrigation, both with stainless steel hand or nickel-titanium rotary files. The root canal remained slightly infected after chemo mechanical preparation in both groups.