In this study we evaluate the potential environmental and health impact of dairy cattle livestock and manure management in the Czech Republic. We present a new approach for national assessments of the environmental impact of an agricultural sector. Emission estimates are combined with a country-specific set of indicators to assess the environmental impact in nine regions with specific environmental characteristics. We estimate the contribution of emissions of ammonia (NH3) and nitrogen oxides (NO) to acidification and terrestrial eutrophication, nitrate (NO3) and phosphate (PO4) to aquatic eutrophication, nitrogen oxides (NO), particulate matter (PM10) and (PM2.5) to human toxicity and methane (CH4) and nitrous oxide (NO) to global warming. We present large regional differences in the environmental and health impact per unit of agricultural production. The regional acidifying, eutrophying and global warming impact of dairy cattle is calculated to be up to three times the national average, depending on the dairy cattle intensity. Aquatic eutrophication is found to be a problem in regions with relatively high eutrophying emissions per hectare of so-called nitrate vulnerable zones. Human toxicity problems caused by dairy cattle livestock and manure management are problematic in regions with a high population density in rural areas. The strength of our approach is the use of country-specific characterisation factors to assess the potential environmental and health impact of agriculture at the sub-national scale. We were able to analyse the potential environmental impact without explicit quantification of specific effects on humans and ecosystems. The results can be used to identify the most polluted areas as well as appropriate targets for emission reduction.