Background: This study aims to assess the effect of root canal disinfection with a 980 µm diode laser following chemomechanical root canal preparation on the severity of pain after root canal treatment (RCT).Methods: In present study, asymptomatic, single-rooted teeth with periapical index (PAI) score 3 or 4 were included. All patients were treated with two visits of root canal treatment including dressing with calcium hydroxide. Patients were randomly divided into two groups (n: 28). ‘Control (no laser)’: The final irrigation was performed using 5ml 2.5% NaOCI, followed by 5 ml 17% EDTA and 5 ml distilled water. ‘Laser Disinfection (LD)’: Root canals were irradiated with 980 µm diode laser after final irrigation at both visits. The pain levels were evaluated using visual analog scale (VAS) after 8, 24, 48 hours and 7 days. In addition, analgesic intake and time intervals were recorded by patients. The collected data were statistically analyzed with the Chi-square and Mann-Whitney U test (p<0.05).Results: The average pain level in the control group after 24 hours was significantly higher than that in the laser group after the first appointment (p<0.05). The average pain level after the second appointment was statistically higher at 24 h and 48 h in the control group (p<0.05). PP levels at 24-hs after the first appointment were higher than the second appointment only in the control group (p<0.05). After first appointment, analgesic use in the control group at 8 h (40%) and 24 h (23%) were significantly higher than LD group (p<0.05).Conclusions: Root canal disinfection with diode laser may reduce PP in single-rooted teeth with PAI score 3 or 4.Trial registration: Effect of the Diode Laser on Post-operative Pain After Endodontic Treatment in Teeth with Apical Periodontitis: NCT04486196. Registered 24 July 2020 - Retrospectively registered, http://clinicaltrials.gov/ct2/show/NCT04486196Clinical Trials ID: NCT04486196