Species richness, composition and abundance of the bryophyte diaspore bank of Central European temperate mixed forests were compared with the forest-floor bryophyte assemblage. The impact of environmental variables and anthropogenic disturbances, including tree species composition, stand structure, microclimate, light conditions, soil and litter properties, management history, and landscape properties, potentially influencing bryophyte diaspore bank assemblages were explored. Thirty-four, 70–100 years old mixed stands with differing tree species composition were examined in the Őrség National Park, Western Hungary. The diaspore bank was studied by soil collection and cultivation, and data were analysed by multivariate methods. Contrary to the forest-floor bryophyte assemblage, where substrate availability, tree species composition and stand structure were the most influential environmental variables, the composition and abundance of the diaspore bank was mainly affected by site conditions (microclimate, litter and soil properties). Species richness of the bryophyte diaspore bank was lower than that of the forest-floor bryophyte assemblage. Short-lived mosses (colonists, short-lived shuttles) were dominant in the diaspore bank, as opposed to the forest-floor bryophyte community, where perennial mosses dominated. In the studied forests, the importance of the bryophyte diaspore bank was relatively low in the regeneration and maintenance of the forest-floor bryophyte vegetation.