The aim of this study was to examine the effect of ozone gas on the remaining bacteria after chemomechanical instrumentation of tooth root canal. The study was carried out at the Department of Endodontics and Restorative dentistry, School of Dental Medicine, University of Zagreb. A total of 37 tooth root canals from 23 teeth (10 incisors, 2 canines, 8 premolars and 3 molars) with a diagnosis of chronic apical periodontitis (17 untreated teeth and 6 retreatments) from 20 adult patients (11 females and 9 male) were selected. Endodontic samples consisted of 74 swabs from 37 canals. The first root canal swab was taken following a completed chemomechanical instrumentation by a sterile paper point after rinsing the root canal with a sterile saline solution. The canal was dried and treated with ozone gas for 40 seconds (HealOzone, Kavo, Germany). After the ozone treatment the canal was rinsed with a sterile saline solution a second swab was taken. The swabs were stored in transport media until cultivation. Microbiological identification was performed by macromorphological, micromorphological, commercial biochemical test microbiological analysis and bacteria count. A significant decrease in the number of bacteria (p < 0.001) was found after the ozone treatment: the total number of bacteria was 82%, 67% of aerobic and 93% of anaerobic bacteria. When analysing individually, a significant decrease was found for Streptococcus mitis and Propionibacterium acnes (p < 0.05). The results of this study shows the efficacy of ozone on the bacterial count reduction in the root canal treatment.