This clinical study reports on the antibacterial effects and outcome of endodontic treatment using either a single-file or a multifile system, associated with calcium hydroxide interappointment medication. The root canals of single-rooted teeth with apical periodontitis were treated by using either Reciproc or BioRaCe instrument systems, 2.5% NaOCl irrigation, and calcium hydroxide medication. Bacteriological samples taken before preparation and immediately before obturation were evaluated for total bacterial counts by quantitative real-time polymerase chain reaction (qPCR). Patients were followed up and the treatment outcome was assessed by clinical and radiographic criteria. Decreasing lesions were classified as success in a lenient criterion or failure in a rigid one. Bacteria were detected in all initial samples (47 cases) and were significantly reduced after treatment in both groups (p<0.001). In the Reciproc and BioRaCe groups, 7/25 (28%) and 11/22 (50%) root canals yielded negative qPCR results before obturation, respectively (p>0.05). Quantitative bacterial reduction was similar between groups (p>0.05). The success rate in the BioRaCe group was 95.5% and 77% in the loose and rigid criterion, respectively. In the Reciproc group, corresponding figures were 88% and 76%. Differences in outcome were not significant (p>0.05). No diseased case showed negative qPCR results for bacteria. A difference of >1 Log10 counts was observed between healed and diseased cases. Root canal treatments of teeth with apical periodontitis using a single-file or a multifile system for preparation, associated with NaOCl irrigation and calcium hydroxide interappointment medication, showed similar antibacterial effectiveness and success rate.