In this single-blinded, prospective, randomized, controlled clinical trial, the effectiveness of an ozone gas or NaOCl/CHX disinfection protocol was compared within the root canal treatment of apical periodontitis. Sixty permanent teeth were randomly allocated to one technique. The clinical/radiographic assessment criteria included clinical symptoms, periapical index (PAI), and size of the apical lesion. In both groups, the root canal was mechanically cleaned and irrigated with NaCl and EDTA. Ozone gas (32gm-3) or NaOCl (3%) was applied followed by a 1-week inter-appointment dressing (Ca(OH)2). As final disinfection, ozone gas (ozone group) or CHX 2% (NaOCl group) was applied. Microbial samples were taken after preparing the access cavity, after chemo-mechanical treatment and after inter-appointment dressing by sterile paper points. Microbial identification was performed by mass spectroscopy (MALDI-TOF-MS) and 16S-rRNA gene sequencing. The treated teeth were blindly re-evaluated after 6/12months. Success rates, the decrease in PAI, the size of apical lesions and bacterial reduction were compared between groups (Fischer's exact test, Mann-Whitney U test). There were no significant differences between the success rates (ozone group: 96.2/95.5% after 6/12months; NaOCl group: 95.5/95.2% after 6/12months). The differences in the decreases in PAI values and apical lesion sizes were also insignificant after 6 and 12months. The bacterial reduction showed no significant differences between groups after chemo-mechanical treatment and after inter-appointment dressing. The most commonly found bacterial genera were Streptococcus spp., Parvimonas spp. and Prevotella spp. The here used ozone gas and NaOCl/CHX protocols showed no difference in bacterial reduction in the sampled areas of the root canals. Within the limitations of the study, ozone gas seems to be a possible alternative disinfection agent within the root canal treatment of apical periodontitis.