ObjectivesTo date, scarce evidence exists around the application of subgingival air-polishing during treatment of severe periodontitis. The aim of this study was to evaluate the effect on the health-related and periodontitis-related subgingival microbiome of air-polishing during non-surgical treatment of deep bleeding pockets in stage III–IV periodontitis patients.Materials and methodsForty patients with stage III–IV periodontitis were selected, and pockets with probing depth (PD) 5–9 mm and bleeding on probing were selected as experimental sites. All patients underwent a full-mouth session of erythritol powder supragingival air-polishing and ultrasonic instrumentation. Test group received additional subgingival air-polishing at experimental sites. Subgingival microbial samples were taken from the maxillary experimental site showing the deepest PD at baseline. Primary outcome of the first part of the present study was the 3-month change in the number of experimental sites. Additional analysis of periodontal pathogens and other sub-gingival plaque bacteria sampled at one experimental site at baseline and 3 months following treatment was performed through a real-time quantitative PCR microarray.ResultsIn the test group, a statistical increase of some health-related species was observed (Abiotropha defectiva, Capnocytophaga sputigena, and Lautropia mirabilis), together with the decrease of pathogens such as ofActinomyces israelii,Catonella morbi,Filifactor alocis,Porphyromonas endodontalis,Sele-nomonas sputigena,Tannerella forsythia,Treponema denticola, andTreponema socranskii. In the control group, statistical significance was found only in the decrease ofFilifactor alocis,Tannerella forsythia, andTreponema socranskii.ConclusionsThe addition of erythritol-chlorhexidine powder seems to cause a shift of the periodontal micro-biome toward a more eubiotic condition compared to a conventional treatment. The study was registered on Clinical Trials.gov (NCT04264624).Clinical relevanceSubgingival air-polishing could help re-establishing a eubiotic microbioma in deep bleeding periodontal pockets after initial non-surgical treatment.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00784-022-04811-4.