To evaluate a new N-monitoring program in the framework of the UN-ECE ICP-Vegetation program using mosses as bioindicators, 490 moss samples were collected at 220 sites in Austria and analyzed for total N (N content) and δ15N signatures. Within-site variability of N content and δ15N signatures was tested for the first time on a large scale and was extremely low compared to between-site variability. N content in moss tissue ranged between 0.76% and 1.99% and δ15N signatures between −10.04 and −2.45. Altitude was significantly correlated with N content (P = 0.021) and δ15N signatures (P < 0.001). When comparing moss data to deposition data from 35 measurement sites, significant correlations between N content and N deposition (P = 0.014) were found. Increasing δ15N signatures provided evidence for a change in N source and its respective isotopic composition with altitude, e.g., due to long-distance transport of reactive N or as a result of changes in the wet:dry deposition ratio. Our study underlines that N deposition can generally be estimated by N content in mosses on a large scale, but that this approach has certain limitations, especially in areas with large differences in altitude and precipitation.