Background Dietary inorganic nitrate (NO3−) and its reduced forms nitrite (NO2−) and nitric oxide (NO), respectively, are of critical importance for host defense in the oral cavity. High concentrations of salivary nitrate are linked to a lower prevalence of caries due to growth inhibition of cariogenic bacteria. Objective In-vitro studies suggest that the formation of antimicrobial NO results in an increase of the pH preventing erosion of tooth enamel. The purpose of this study was to prove this effect in-vivo. Methods In a randomized clinical study with 46 subjects we investigated whether NO3− rich beetroot juice exhibits a protective effect against caries by an increase of salivary pH. Results Our results show that, in comparison to a placebo group, consumption of beetroot juice that contains 4000 mg/L NO3− results in elevated levels of salivary NO2−, nitrite NO3−, and NO. Furthermore, we determined an increase of the mean pH of saliva from 7.0 to 7.5, confirming the anti-cariogenic effect of the used NO3−-rich beetroot juice. Conclusions Taken together, we have found that NO3−-rich beetroot juice holds potential effects against dental caries by preventing acidification of human saliva. Trial registration C-87-15 (Ethics Commissions of Upper Austria).