Background: Third‐generation carbon dioxide (3‐CO2) lasers have handpieces that accommodate a periodontal insert that permits irradiation directly into the periodontal pocket/sulcus. The purpose of this pilot study was to evaluate, by scanning electron microscopy (SEM), the surface effects of 3‐CO2 laser treatment on the root surfaces and soft tissues and to investigate the effects of 3‐CO2 laser on periodontal pathogenic bacteria compared to negative controls. Methods: Five patients with chronic periodontitis and treatment planned for a maxillary immediate denture were included in the study. Periodontal pockets with a mean probing depth of 5.5 ± 0.8 mm were irradiated one time by CO2 laser (wavelength of 10,600 nm) at a power of 2.2 W, 50 Hz, an 80‐millisecond pulse length, and an exposure rate of 1 mm per 5 seconds. DNA analysis of eight periodontal bacteria was performed on samples collected from laser‐treated and control sites prior to and immediately following treatment and was compared between groups. In addition, block biopsies, including soft tissue, were harvested for SEM examination. Results: SEM examination of test group specimens showed heat damage on the soft tissues in three of 17 specimens (17.6%). In addition, 11.7% (two of 17) of the 3‐CO2–treated teeth exhibited localized slight damage to root surfaces. Microbiologic results of the control sites indicated that 90.6% of the bacterial counts remained the same, 6% increased, and 3% decreased. In the test group, 71.25% of the bacterial count analyses for the eight different periodontal microbes remained the same, 12.50% increased, and 16.25% decreased. Conclusion: A one‐time use of the 3‐CO2 laser in periodontal pockets did not sterilize or substantially reduce subgingival bacterial populations compared to negative controls.