Nitrogen (N) fixation in the feather moss-cyanobacteria association represents a major N source in boreal forests which experience low levels of N deposition; however, little is known about the effects of anthropogenic N inputs on the rate of fixation of atmospheric N in mosses and the succeeding effects on soil nutrient concentrations and microbial community composition. We collected soil samples and moss shoots of Pleurozium schreberi at six distances along busy and remote roads in northern Sweden to assess the influence of road-derived N inputs on N fixation in moss, soil nutrient concentrations and microbial communities. Soil nutrients were similar between busy and remote roads; N fixation was higher in mosses along the remote roads than along the busy roads and increased with increasing distance from busy roads up to rates of N fixation similar to remote roads. Throughfall N was higher in sites adjacent to the busy roads but showed no distance effect. Soil microbial phospholipid fatty acid (PLFA) composition exhibited a weak pattern regarding road type. Concentrations of bacterial and total PLFAs decreased with increasing distance from busy roads, whereas fungal PLFAs showed no distance effect. Our results show that N fixation in feather mosses is highly affected by N deposition, here derived from roads in northern Sweden. Moreover, as other measured factors showed only weak differences between the road types, atmospheric N fixation in feather mosses represents a highly sensitive indicator for increased N loads to natural systems.