Large herbivores are keystone species in many forest areas, as they shape the structure, species diversity and functioning of those ecosystems. The European bison Bison bonasus has been successfully restored after extinction in the wild at the beginning of 20th century. As free-ranging populations of the species were re-established mainly in forest habitats, knowledge of the impact by the largest European terrestrial mammal on tree stands is essential. This helps to make management and conservation decisions for viable population maintenance of the species in the wild. Using a novel DNA-based method of herbivore diet analysis, the trnL approach (DNA-barcoding), we investigated the influence of different foraging conditions (access to supplementary fodder) on bison diet in winter and its potential impact on woody species. Faecal samples were collected from different bison treatment groups: (1) intensively fed; (2) less intensively fed; (3) non-fed utilising forest habitats; and (4) non-fed utilising agricultural areas surrounding the Forest. These were analysed to estimate the proportion of different plant groups consumed by bison. Bison groups differed significantly in their diet. The amount of woody materials (trees and shrubs) consumed by bison increased with decreasing access to supplementary fodder, ranging from 16% in intensively fed bison to 65% in non-fed bison utilising forest habitats. Inversely, the amount of herbs, grasses and sedges decreased from 82% in intensively fed bison to 32% in non fed bison utilising forest habitats. The species of trees mainly browsed by bison, Carpinus/Corylus, Betula sp. and Salix sp., were of lower economic importance for forest management. The impact of bison on tree species needs further investigation, however, we can predict that browsing by bison, mainly on Carpinus/Corylus, makes an insignificant impact on forestry due to the high and increasing representation of this species in the forest understory. Supplementary feeding has several negative effects on bison ecology and health, therefore reduced and distributed supplementary feeding should be applied as the management practice in the Białowieża Forest.Research highlights▶ Impact of large ungulates on tree stands essential to make management decisions. ▶ Use of novel DNA-based method to analyse diet of herbivores. ▶ Habitat and supplementary feeding influence European bison diet. ▶ Proportion of woody material in bison diet increase with decreasing supplementary feeding. ▶ Browsed by bison tree species has low importance for forest management.