► N2 fixation in forest mosses was studied using acetylene reduction and 15N2 methods. ► The methods gave corresponding results with the conversion factor of 3.3. ► Moss-associated N2 fixation was higher in the northern samples with low N deposition. ► Methanotrophic activity associated with forest mosses was low. Nitrogen deposition has decreased the plant-associated nitrogen (N2) fixation when measured using the indirect acetylene reduction assay (ARA). However, nitrogen deposition can also lead to changes in the diversity of moss symbionts, e.g. affect methanotrophic N2 fixation, which is not measured by ARA. To test this hypothesis we compared ARA with the direct stable isotope method (15N2 incorporation) and studied methanotrophy in two mosses, Hylocomium splendens and Pleurozium schreberi, collected from seven forest sites along a boreal latitudinal N deposition transect. We recognized that the two independent N2 fixation measures gave corresponding results with the conversion factor of 3.3, but the 15N2 method was more sensitive for finding a signal of low N2 fixation activity. Methane carbon fixation associated with mosses was under the detection limit (<2nmolCg−1h−1). N2 fixation rates were more pronounced in the mosses with higher C/N ratio, and in the green upper parts of the shoot than in the lower brownish parts. Sequencing of nifH genes revealed that dominating diazotrophs were affiliated to cyanobacterial genera Nostoc and Nodularia, but methanotrophic diazotrophs were not found in the nifH libraries. We conclude that the suppression of N2 fixation along the deposition gradient was consistent regardless of the measurement technique, and microbial community changes toward methanotrophic or otherwise acetylene-sensitive N2 fixation could not explain this trend.