The aim of this study was to evaluate the clinical, anti-inflammatory, and osteoimmunological benefits of the single (PT) and repeated laser phototherapy (rPT) as an adjunctive treatment of inflamed periodontal tissue. Twenty-seven patients with chronic periodontitis were randomly divided into three groups of nine patients each in order to undergo scaling and root planing (SRP), SRP followed by one session of adjunctive PT (Day 1; SRP + PT), or SRP followed by adjunctive repeated PT five times in 2 weeks (Days 1, 2, 4, 7, and 11; SRP + rPT). For phototherapy session, a diode laser (λ = 670 nm, 200 mW, 60 s/tooth) was applied into the sulcus. Clinical parameters, including full-mouth plaque score, full-mouth bleeding score, probing pocket depth, and clinical attachment level were recorded. Samples of gingival crevicular fluid (GCF) were taken at baseline, 4, and 8 weeks after treatment. Interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), receptor activator of nuclear factor κΒ ligand (RANKL), and osteoprotegerin (OPG) levels in the collected GCF were measured. PT used in a single or repeated doses, does not produce a significant reduction in the clinical parameters essayed (p > 0.05). Levels of IL-1β in GCF were significantly reduced in SRP + PT and SRP + rPT groups compared with the SRP group (p < 0.05). However, the SRP + rPT group showed a significant reduction of pro-inflammatory cytokine TNF-α and RANKL/OPG ratio at 4 weeks post-treatment compared with the SRP + PT and SRP groups (p < 0.05). SRP + PT group also showed a significant reduction in TNF-α and RANKL/OPG ratio at 8 weeks post-treatment compared with the SRP group (p < 0.05). PT exerts a biostimulative effect on the periodontal tissue. Multiple sessions of PT showed a faster and greater tendency to reduce proinflammatory mediators and RANKL/OPG ratio.