In this paper, we assess and map the risk that atmospheric nitrogen (atN) pollution poses to biodiversity in Natura 2000 sites in mainland Portugal. We first review the ecological impacts of atN pollution on terrestrial ecosystems, focusing on the biodiversity of Natura 2000 sites. These nature protection sites, especially those located within the Mediterranean Basin, are under-characterized regarding the risk posed by atN pollution. We focus on ammonia (NH3) because this N form is mostly associated with agriculture, which co-occurs at or in the immediate vicinity of most areas of conservation interest in Portugal. We produce a risk map integrating NH3 emissions and the susceptibility of Natura 2000 sites to atN pollution, ranking habitat sensitivity to atN pollution using expert knowledge from a panel of Portuguese ecological and habitat experts. Peats, mires, bogs, and similar acidic and oligotrophic habitats within Natura 2000 sites (most located in the northern mountains) were assessed to have the highest relative risk of biodiversity change due to atN pollution, whereas Natura 2000 sites in the Atlantic and Mediterranean climate zone (coastal, tidal, and scrubland habitats) were deemed the least sensitive. Overall, results allowed us to rank all Natura 2000 sites in mainland Portugal in order of evaluated risk posed by atN pollution. The approach is of great relevance for stakeholders in different countries to help prioritize site protection and to define research priorities. This is especially relevant in countries with a lack of expertise to assess the impacts of nitrogen on biodiversity and can represent an important step up from current knowledge in such countries.