Moss-associated nitrogen (N₂) fixation performed by epiphytic, N₂-fixing bacteria (diazotrophs) contributes significantly to ecosystem N input in pristine habitats. While we have some understanding of the effects of climate warming on moss-associated N₂ fixation, we lack data on effects of freeze-thaw cycles (FTCs) on diazotroph activity, although increased frequency of FTCs is predicted. We collected the widespread moss Pleurozium schreberi along a climate gradient (temperate, boreal, arctic) and exposed moss and associated diazotrophs to severe (20 °C difference, cycling between +10 and −10 °C) and mild (6 °C difference, ±3 °C) diurnal FTCs. We measured N₂ fixation in mosses over 8 weeks and assessed their nutrient loss (fixed N₂, total dissolved N, ammonium, phosphate) during the FTCs. We expected lower nitrogenase activity in mosses exposed to more severe FTCs and different sensitivities of N₂ fixation towards FTCs along the climate gradient. However, no differences were found in N₂ fixation between mild and severe FTCs, but N₂ fixation in mosses from the temperate heath was less susceptible to FTCs than those from colder sites, suggesting adapted temperate diazotroph communities. Mosses lost little N, most at constant, positive temperatures, while more phosphate was lost from mosses exposed to FTCs, depending on the positioning along the climate gradient, mirroring nutrient demand and limitation. Our results show that moss-associated N₂ fixation is less susceptible towards FTCs than expected but nutrient loss from moss carpets can increase following FTCs, with consequences for nutrient pools and fluxes.