Objectives The aim of this pilot study was to evaluate the clinical and microbiological outcomes of light-activated disinfection (LAD) alone or combined with probiotics as an adjunct to non-surgical periodontal treatment. Materials and methods In this single-blinded, randomized, controlled clinical pilot study, 48 patients (28 females and 20 males) with untreated periodontitis (stages II and III, grade B) were included. Using a parallel-group design, patients were randomly assigned into 3 groups to receive subgingival debridement (SD) alone (group 1, n = 16), SD with LAD (group 2, n = 16), or SD with LAD plus probiotic treatment (group 3, n = 16). Probing pocket depth (PPD), clinical attachment level (CAL), bleeding on probing (BOP), gingiva-index simplified (GIs), plaque-control record (PCR), and subgingival microbiological samples were analyzed at baseline, 3 months, and 6 months of follow-up. Results All treatment modalities demonstrated clinical improvements in PPD and CAL at 6 months compared to baseline but without a statistical significant difference between the groups. The combination of SD + LAD + probiotic treatment (group 3) demonstrated significantly greater reductions in BOP, GIs, and red complex bacteria P. gingivalis and T. forsythia compared with other groups at 6 months (p < 0.05). Conclusions A single application of LAD as an adjunct to SD provided no additional clinical and microbiological benefits compared to SD alone. The combination of SD + LAD + probiotic treatment in group 3 led to further improvements of the inflammatory parameters. Clinical relevance The additional use of probiotics in periodontal treatment can be a useful approach to support inflammation and infection control of periodontal tissues. Further studies are necessary to determine the extent of added benefit for this treatment approach.