The investigations of flora and fauna in Protected Areas provide important contributions to the knowledge on the status of unmanaged natural ecosystems. In 2017–2019, we studied the vascular plant flora of Behali Reserve Forest (Assam, India). The reserve forest covers 140.16 km2 area of semi-evergreen forest. We investigated the taxonomic composition of the flora and diversity of growth forms of species and habitats. In addition, we conducted global IUCN Red List assessment of two endemic species, Aristolochia assamica and Chlorophytum assamicum. We found that the vascular plant flora of Behali Reserve Forest consists of 281 taxa, including 272 species, one subspecies and eight varieties. They belong to 206 genera and 79 families. Among them, Pandanus unguifer was recorded for the first time for the flora of Assam. In growth form spectrum of vascular plants, trees, vines and shrubs predominated, followed by epiphytes, forbs or herbs, geophytes, annuals and parasites. The largest number of vascular plants was found in dense forests, while open habitats were characterized by the smallest species richness. Chlorophytum assamicum is assessed as CR B1ab(iii)+2ab(iii); D, and Aristolochia assamica as EN B2ab(iii). At the same time, there is a threat of reduction or disappearance of their populations. This is especially crucial for C. assamicum, represented by a single world population. Undoubtedly, further investigations of flora could result in an increase in number of the vascular plant flora of Behali Reserve Forest, as well as providing essential insights into population trends of several other species as per IUCN Red List categories and criteria.