Objectives In this clinical study, we aim to evaluate the effectiveness of non-thermal atmospheric pressure plasma (NAPP), which is a novel procedure used in periodontal pocket decontamination adjunctive to non-surgical periodontal treatment (NSPT). Methods The study included 25 systemically healthy periodontitis patients. In the split-mouth design, NAPP application into the pockets, in addition to NSPT, was performed. Clinical periodontal data, gingival crevicular fluid, and subgingival plaque samples of patients were taken before and during the first and third months of treatment. Biochemical assays were conducted using enzyme-linked immunosorbent assay. Analysis of bacteria was performed with polymerase chain reaction method. Results There was more clinical attachment level (CAL) gain in the 3rd month in the test group (deep pockets: 3.90 mm, pockets >= 5 mm: 2.72 mm) compared to the control group (deep pockets: 3.40 mm, pockets >= 5 mm: 2.58 mm) (p < 0.05), but no significant difference between groups in CAL. Clinical periodontal parameters improved in both study groups (p < 0.05). However, the gingival index (GI) and the bleeding on probing (BOP) rate decreased more in the test group (GI: 0.55, BOP: 9.48%, and GI: 0.38, BOP: 8.46% in the 1st and 3rd months, respectively) compared to the control group (GI: 0.68, BOP: 13.43%, and GI: 0.52, BOP: 14.58%) (p < 0.05). In addition, there was no significant difference in probing depth and biochemical markers between groups (p > 0.05). It was observed that NAPP reduced the number of bacteria more than the control group in the 1st and 3rd months. Conclusions It was seen that the single-time NAPP application concurrent with NSPT provided additional CAL gain, elimination of putative periodontopathogens and reduced their recolonization. Longitudinal studies with larger population and longer time are required.