Fast-growing hybrid aspen has been widely planted on former agricultural lands in Northern and Eastern Europe to produce pulpwood and sequester carbon into woody biomass. The biodiversity of mature hybrid aspen plantations has so far been rarely analysed. Moreover, the potential of hybrid aspen plantations to host flora typical of European aspen stands – recognised as biodiversity hotspots in Eurasian boreal forests – has not been evaluated. This study focused on two organism groups with contrasting habitat preferences – ground-dwelling herbs and epiphytic lichens. We sought to clarify how environmental and landscape factors influenced the diversity of herbs, lichens, and species characteristic of European aspen stands (EA species). Data were collected across 42 study plots within 20 hybrid aspen plantations in Estonia. Altogether, 162 herb species and 65 lichens were recorded, including 92 species also found in European aspen stands. The proportion of EA species was higher among lichens (86.2 %) than among herbs (22.2 %), reflecting a stronger legacy effect of former agricultural land-use on ground-dwelling species than on epiphytes. Responses of herb and lichen diversity to environmental variables differed. Richness of herbs was related to litter and stand characteristics. Compositional analyses also highlighted the role of soil-litter variables and stand structure in herb composition. Lichens responded to landscape-related variables. A greater extent of older forests around the study plots increased the richness of EA species. In conclusion, mature hybrid aspen plantations are relatively species-rich and can enhance landscape connectivity for forest species, particularly epiphytes, in agricultural landscapes. Keywords: Lichens; Herbs; Short-rotation forestry; Biodiversity; Populus.