The study aim was to determine high frequency diathermocoagulation operation modes safe for periodontal tissues and providing the best antimicrobial effect in the root canal system. In vitro study was conducted on 12 extracted upper and lower incisors. High frequency monopolar diathermocoagulation device DK-35MS (2640 kHz; 1.5 kΩ) in six operation modes was used. Root surface heating degree was measured with compact infrared radiation converter (thermal imager Seek Thermal) and distance infrared thermometer Testo 830-T1. After determination of safe operation modes antibacterial effect was assessed. The strains of Streptococcus sanguis, Streptococcus mutans, Enterococcus faecalis, Escherichia coli and Candida albicans were obtained from root canals with inflamed pulp. Results: for endodontic diathermocoagulation procedure the safe operation modes (for the tissues surrounding the tooth) were #3, #4 and #6 with the capacity 3.06—5.4 W for 3 seconds. In operation modes #3 and #6 with the power 5.4 W and 4.1 W, respectively, the best antimicrobial effect was achieved against the studied microorganisms. Conclusion: High frequency monopolar diathermocoagulation can be used in endodontic treatment. The recommended power modes of diathermocoagulation in root canals are 4.1 W and 5.4 W, the recommended time is 3 seconds. These operation modes do not cause root surface heating and provide antimicrobial effect against endodontic pathogens.