Aim: Identify bacterial species in necrotic root canals of teeth with chronic apical periodontitis, and to evaluate the antimicrobial effectiveness of rotary and reciprocating instrumentation techniques in the elimination of bacterial species from necrotic canals of teeth with chronic apical periodontitis. Materials and methods: Forty patients with necrotic teeth with chronic apical periodontitis were selected for this study based on previously defined inclusion and exclusion criteria. After they had signed an informed consent, the participants were included in this clinical study and randomly assigned to one of two experimental groups depending on the root canal instrumentation technique used: Group 1. Wave One Gold technique; Group 2. ProTaper Next instrumentation technique. In both groups, root canals were irrigated with 2.5% sodium hypochlorite (NaOCl) solution during the instrumentation, and then the final irrigation protocol was applied in both groups: 3 ml NaOCl, 2 ml ethylenediaminetetraacetic acid (EDTA) and 2 ml NaOCl. A single visit root canal treatment was performed by one doctor of dental medicine, who finished intracanal procedures by the cold lateral condensation technique using an epoxy resin based sealer and gutta-percha. Microbiological samples from the root canal were collected for three times using a paper point technique: before instrumentation (sample 1.), after chemomechanical treatment (sample 2.) and after the final irrigation protocol (sample 3.). Microbiological analysis was carried out by identification of bacteria using MALDI-TOF mass spectrometry and colony forming units (CFUs) counting by the culture method. The Wilcoxon and chi-quadrat test were used for statistical analysis with a significance level of 0.05. Results: The first sample showed the presence of aerobic and anaerobic bacterial species in the patients. After chemo-mechanical treatment of root canals with Wave One Gold and ProTaper Next technique, the number of bacteria was significantly reduced in both groups (p<0.001). The final irrigation protocol also further reduced the number of bacterial species in both groups (p<0.001). The ProTaper Next technique and Wave One Gold technique were similarly successful in reducing the number of bacterial colonies (p>0,05).