Objectives <p>Nd: YAG laser, a near-infrared laser, offers advantages such as sterilization, hemostasis, and tissue ablation, making it a promising adjunctive tool for minimally invasive periodontal treatment. This study aimed to evaluate the clinical efficacy of Nd: YAG laser-assisted nonsurgical treatment in stage III–IV periodontitis and its impact on the subgingival microbial community structure using 16&#xa0;S rRNA sequencing.</p> Materials and methods <p>A total of 20 patients with stage III-IV periodontitis were randomly divided into a subgingival scaling and root planning (SRP group) (<i>n</i> = 10) and a Nd: YAG laser-assisted SRP group (Nd group) (<i>n</i> = 10). Clinical parameters were compared before and after treatment for 3 months, and subgingival plaque samples were collected for 16&#xa0;S rRNA microbial analysis.</p> Results <p>Both groups exhibited significant improvements in periodontal clinical parameters after treatment (<i>P</i> &lt; 0.01), with greater gains in the Nd group (<i>P</i> &lt; 0.05). Alpha diversity decreased after treatment in both (<i>P</i> &lt; 0.05), more markedly in the Nd group (<i>P</i> &lt; 0.01). Beta diversity analysis showed that significant clustering was formed in the subgingival plaque samples before and after treatment in both groups, with larger microbial shifts in the Nd group(<i>P</i> &lt; 0.05). After treatment, the relationship network was significantly simplified.</p> Conclusions <p>Non-surgical treatment significantly improved periodontal parameters in stage III-IV periodontitis patients, reducing subgingival microbiota diversity and optimizing its composition.</p> Clinical relevance <p>Nd: YAG laser-assisted SRP demonstrated superior clinical outcomes versus SRP alone, with microbiological analysis confirming its adjunctive benefits and enhanced clinical efficacy.</p>