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This longitudinal study demonstrates that non-surgical periodontal therapy (NSPT) in patients with advanced periodontitis results in significant reductions in pathogenic bacterial extracellular vesicles (EVs) in saliva and plaque, alongside increased host cytokines associated with healing. Notably, shifts in fungal EV populations were also observed, suggesting microbial ecosystem modulation. These findings position salivary EVs as promising non-invasive biomarkers for monitoring therapeutic efficacy in periodontitis.