BACKGROUND: Periodontitis is a polymicrobial, multifactorial infection that affects the supporting structures of teeth. Melatonin, a biomolecule with anti-inflammatory, antibacterial, and antioxidant properties, has demonstrated promising results in various medical fields, including dentistry. OBJECTIVE: This study aimed to evaluate the effectiveness of 1% (w/v) melatonin gel as an adjunct to Non-Surgical Periodontal Therapy (NSPT) in improving clinical periodontal parameters, reducing antimicrobial activity against Aggregatibacter actinomycetemcomitans and Prevotella intermedia, and increasing superoxide dismutase (SOD) levels in gingival crevicular fluid (GCF) among patients with stage II periodontitis. METHODS: A split-mouth randomized controlled trial was conducted on 24 periodontitis patients. Two sites per patient were randomly assigned: the test site underwent scaling and root planing (SRP) followed by intra pocket application of 1% melatonin gel, while the control site received SRP alone. Clinical parameters, including the Plaque Index (PI), Gingival Index (GI), Gingival Bleeding Index (GBI), Periodontal Pocket Depth (PPD), and Clinical Attachment Loss (CAL), were assessed at baseline, 1 month, and 3 months. Subgingival plaque samples and GCF were collected to evaluate microbial and biochemical changes. RESULTS: Both groups showed statistically significant improvements in clinical parameters from baseline to the 3rd month post-therapy. A quantitative reduction in Aggregatibacter actinomycetemcomitans and Prevotella intermedia was observed at both sites. Additionally, the test site exhibited a greater increase in SOD levels compared to the control site. CONCLUSION: The adjunctive application of melatonin gel with SRP demonstrated enhanced antioxidant potential and improved clinical outcomes in patients with stage II periodontitis. TRIAL REGISTRATION TRIAL REGISTRY: ISRCTN. Trial registration number ISRCTN40460432. Date of Registration: 22/10/2024. "Retrospectively registered".