ObjectivesThe effects of water flossing on dental plaque removal have been suggested, but its ecological impact on dental plaque microbiota needs further investigation. In addition, whether this plaque control measure by water flossing promotes the control of halitosis still needs clinical validation. The aim of this study was to evaluate the effects of water flossing on gingival inflammation and supragingival plaque microbiota.Materials and methodsSeventy participants with gingivitis were randomly assigned to control (toothbrushing) and experimental (toothbrushing + water flossing) groups (n= 35). Participants were recalled at 4, 8, and 12 weeks, and their gingival index, sulcus bleeding index, bleeding on probing, dental plaque index, and oral malodor values were measured. The microbiota of supragingival plaque was further investigated using 16S rRNA sequencing and qPCR.ResultsSixty-three participants completed all revisits (control:n= 33; experimental:n= 30). The experimental and control groups exhibited similar clinical characteristics and dental plaque microbiota at baseline. Adjunctive water flossing effectively reduced the gingival index and sulcus bleeding index as compared to the toothbrushing control group. The water-flossing group showed reduced oral malodor at week 12 as compared to the baseline. Consistently, the water-flossing group exhibited altered dental plaque microbiota at week 12, characterized by a depletion ofPrevotellaat genus level andPrevotella intermediaat species level as compared to the toothbrushing control. In addition, the plaque microbiota of water-flossing group exhibited a more aerobic phenotype, while the control group was more anaerobic.ConclusionsDaily water flossing can effectively alleviate gingival inflammation and reduce oral malodor, possibly by depleting oral anaerobes and altering the oral microbiota to a more aerobic phenotype.Clinical relevanceWater flossing adjunctive to toothbrushing effectively alleviated gingival inflammation, representing a promising oral hygiene practice to promote oral health.Clinical trial registrationThe trial was registered in the Chinese Clinical Trial Registry (http://www.chictr.org.cn/showprojen.aspx?proj=61797, #ChiCTR2000038508) on September 23, 2020.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00784-023-05081-4.