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In this study, we compared the antimicrobial efficacy of various root canal preparation techniques and instruments in the root canals of 40 human first maxillary premolars extracted for orthodontic reasons. After extirpation of the pulp, teeth were sterilized in an autoclave. Then, the root canals were infected with Enterococcus faecalis suspension and the teeth incubated at 37°C for 24 h. Thereafter, the teeth were divided into five groups: preparation with K-file or K-Nitiflex file (step-back technique; groups 1 and 2), K-reamer (standardization technique; group 3), irrigation only (group 4), and no treatment (group 5). Before and after treatments, samples were taken for culture. Results were evaluated after 24-h incubation in E67 selective culture medium. We observed a considerable reduction in bacterial population after mechanical preparation. No significant differences were seen among the efficacy of the various preparation techniques and files used.