The present study aimed to evaluate the potential hazardous effects of NO3− concentration in drinking water on health, feed intake, rumen fermentation and microbiota, and nitrogen excretion of Holstein heifers fed a high-concentrate diet for a sustainable water use. Twenty-four Holstein heifers were individually allocated and assigned to one of four treatments with increasing drinking water NO3− concentration: CTR, without NO3−; LOW, with 44 mg NO3−/L; MOD, with 110 mg NO3−/L; and HIGH, with 220 mg NO3−/L. The entire study lasted 168 days. Fortnightly water NO3− concentration and daily feed and water intake were recorded. Blood parameters, rumen pH, volatile fatty acids, NO3− and NO2− concentration, microbiota, and apparent total tract digestibility were determined at the beginning and at the end of the study. Most of the analyzed parameters were similar among treatments. Denitrifying bacteria population, estimated as nosZ gene copies, were greater in HIGH animals than in CTR animals at the end of the study. In conclusion, drinking water NO3− concentration up to 220 mg/L has no detrimental effect on health, feed intake, rumen fermentation, nor N excretion in dairy beef cattle for periods up to 168 days; moreover, denitrifying bacteria population increased, which are related with the neutralization of the greenhouse gas N2O.