To compare the efficacy of combined treatment of Er:YAG laser (ERL) and low-level laser therapy (LLLT) with single laser applications, and scaling and root planing (SRP) for non-surgical periodontal treatment. In a randomized controlled trial, 25 non-smoking Stage II or Stage III periodontitis patients were recruited. The four intraoral quadrants were randomly assigned to four different treatments: (1) combined application with ERL plus SRP plus LLLT; (2) ERL plus SRP; (3) SRP plus LLLT; and (4) SRP. We assessed periodontal indexes, including probing depth (PD), clinical attachment level (CAL), bleeding index (BI), and plaque index (PLI), along with three cytokines (IL-1β, TNF-α, IL-10) from gingival crevicular fluid and red complex pathogens from subgingival dental plaque at baseline, 3 months, and 6 months. For initial moderate pockets (4mm≤PD≤6mm), quadrants treated with ERL+SRP+LLLT, ERL+SRP, and SRP+LLLT exhibited greater PD improvement compared to the control (SRP) quadrants at the 3-month follow-up (1.25±1.06, 1.23±1.12, 1.00±1.21vs. 0.98±1.21mm) and the 6-month follow-up (1.35±1.06, 1.23±1.17, 1.35±0.98vs. 0.98±1.23mm) (p=0.002). Quadrants treated with ERL+SRP+LLLT and SRP+LLLT showed more CAL gain means than the control quadrants at the 3-month follow-up (0.96±1.42, 0.61±1.39vs. 0.55±1.57mm) and the 6-month follow-up (0.84±1.54, 0.89±1.49vs. 0.48±1.68mm) (p=0.008). For initial deep pockets (PD≥7mm), the ERL+SRP+LLLT quadrants had more PD improvement and CAL gain compared to the control quadrants at follow-up. There were no significant differences in BI, PLI, inflammatory cytokines, and periodontal pathogens among the four groups. The combined application of ERL and LLLT demonstrated potential efficacy in reducing PD, particularly for deep pockets. To compare the therapy effect of combined use of Er:YAG laser (ERL) and low level laser therapy (LLLT) with single laser applications, and traditional periodontal treatment (SRP). A total of 25 nonsmoking patients with periodontitis were involved, and their mouths were divided into four sections, each receiving a different treatment: ERL+SRP+LLLT, ERL+SRP, SRP+LLLT, and SRP. Clinical indexes and laboratory indicators were assessed at baseline, 3 months, and 6 months. After six months, for initial moderate pockets, combined laser group and single laser group showed better improvements than traditional group in reducing the depth of periodontal pockets and increasing attachment levels. But for initial deep pockets, only combined laser group showed better improvement than traditional group. There were no significant differences in bleeding, plaque, inflammation, or harmful bacterial levels among the groups. These findings suggest that the integration of Er:YAG laser and lowlevel laser therapy into standard periodontal treatment may enhance the treatment's benefits in reducing pocket depth, especially for severe conditions.