Treatment of severe periodontitis with non-surgical therapy remains challenging in dentistry. The present study aimed to evaluate the clinical efficacy of hydrogen peroxide (H2O2) photolysis-based antimicrobial chemotherapy adjunctively performed with root debridement (RD) for moderate to severe periodontitis. A randomized controlled trial was conducted that included 53 patients with 142 test teeth. The test teeth were randomly assigned to one of three treatment groups: Group 1, RD + H2O2 photolysis; Group 2, RD followed by administration of a local drug delivery system (minocycline chloride gel); or Group 3, RD alone. Clinical and microbiological examination were performed for up to 12 weeks following treatment. Probing pocket depth (PPD) and bleeding on probing (BoP) were improved after each treatment session. At 12 weeks, Group 1 had achieved significantly lower PPDs than the other groups, though there were no significant differences in BoP between Group 1 and the other groups. Counts of Porphyromonas gingivalis, a known periodontal pathogen, in Group 1 were significantly lower than those in Group 3, and were comparable to those in Group 2. Therefore, it is suggested that H2O2 photolysis treatment can be used as a novel adjunctive antimicrobial chemotherapy for non-surgical periodontal treatment.