The present study aims to evaluate and compare the effects of 1% melatonin gel plus platelet-rich fibrin (PRF) and PRF alone in treatment of furcation defects, clinically and radiographically using cone-beam computed tomography. This split-mouth clinical trial included 23 patients with 46 bilateral Grade II furcation defects in first or second maxillary or mandibular molars. Control group was treated with PRF alone while the test group was treated with 1% melatonin gel and PRF. Clinical attachment level (CAL) and horizontal probing depth (HPD) were primary outcome parameters while secondary outcome parameters were pocket probing depth (PPD) and defect volume (DV) assessed at 3 and 6 months postoperatively. Significantly greater mean reduction of PPD, HPD, and CAL gain was observed in test group (2.23±0.41, 2.36±0.68, and 2.97±0.62mm, respectively) in comparison with control group (2.83±0.78, 2.85±0.67, and 3.21±0.86mm, respectively) at 6 months. A significant reduction in the mean DV was exhibited radiographically in test group (1.06±0.5 mm3) as compared with control group (3.94±1.32 mm3) at the end of 6 months. Both the groups showed improvements in assessed parameters. In Grade II furcation defects the combination therapy of 1% melatonin + PRF shows a statistically significant degree of bone fill within the periodontal tissues and also better results in terms of decrease in PPD, HPD, and a greater CAL gain.