Supragingival air polishing of teeth effectively removes dental plaque and extrinsic stain on coronal tooth surfaces, but its impact on specific periodontal pathogens in adjacent subgingival biofilms is not known. This study assessed the microbiological effect of supragingival air polishing on the subgingival microbiota of individuals with severe periodontitis. Supragingival air polishing with a sodium bicarbonatebased powder was performed on 15 adult test subjects, with the nozzle of the air polishing device aimed apically at a 45° angle onto tooth surfaces immediately coronal to the entrance of periodontal pockets. Supragingival prophylaxis paste polishing, using a slow-speed handpiece, was carried out on 13 adult control subjects. Subgingival specimens were collected from a single 5 mm to 7 mm periodontal pocket with bleeding on probing in each of the study participants before and immediately after supragingival polishing procedures. Viable bacterial counts and selected putative periodontal pathogens (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia/nigrescens, Fusobacterium nucleatum, Parvimonas micra, Campylobacter species) were quantified by microbial culture, and motile morphotypes (spirochetes and motile rods) by phase-contrast microscopy. Statistically significant decreases were detected after supragingival air polishing in total viable counts (84.9% decrease), in P. intermedia/nigrescens, F. nucleatum, Campylobacter species, total proportions of red/orange complex periodontal pathogens (82.3% decrease), and in motile morphotypes (85.3% decrease). No statistically significant subgingival microbiological changes occurred with supragingival prophylaxis paste polishing. Supragingival air polishing of teeth, but not supragingival prophylaxis paste polishing, may serve as a useful therapeutic adjunct to disrupt and help remove pathogenic biofilms in deep periodontal pockets.