Nitrate (NO3−) contained in food and beverages can transiently increase nitric oxide (NO) availability following a stepwise reduction to nitrite (NO2−) by commensal bacteria in the oral cavity. We tested the hypothesis that regular ingestion of dietary NO3− would influence the oral microbiome, the capacity to reduce NO3− to NO2− in saliva, and the vascular responses to an acute dose of NO3−. The abundance of bacterial species on the tongue, the availability of NO markers, and vascular function were assessed in 11 healthy males before and after 7 days of supplementation with NO3−-rich beetroot juice and a NO3−-depleted placebo. As expected, saliva and plasma NO2− and NO3− were significantly elevated after NO3− supplementation (all P < 0.05) but not placebo. We found that NO3− supplementation increased salivary pH (7.13 ± 0.54 to 7.39 ± 0.68, P = 0.043) and altered the abundance of some bacteria previously implicated in NO3− reduction: Neisseria (from 2% ± 3%–9% ± 5%, P < 0.001), Prevotella (from 34% ± 17%–23% ± 11%, P = 0.001) and Actinomyces (from 1% ± 1%–0.5% ± 0.4%). Despite these alterations to the oral microbiota, an acute dose of NO3− increased salivary and plasma NO2−, reduced systolic blood pressure and increased the response to flow mediated dilation to a similar extent before and after 7 days of supplementation (P > 0.05). Our study establishes that supplementing the diet with NO3− for a sustained period can alter the oral environment in favour of health but does not impact the response to an acute NO3− dose. Acute ingestion of NO3− results in transient improvements in vascular function but the dietary induced adaptations to the oral bacteria did not enhance these effects.