Recent concerns to strengthen the role of Tropical Botanic Gardens (TBGs) in conservation are constrained by inadequate representation of models of best practices. This also presents minimal opportunities to explore and fully appreciate the contribution of botanic gardens to biodiversity conservation, especially in urban landscapes. We contribute to addressing this issue using the KNUST Botanic Garden. The study explores biodiversity variation in two microsites (upland and inundated areas), within the context of threats from urbanization. The composition, diversity, and conservation status of plants were assessed from three 50 × 50 m plots each located at the two microsites. A total of 78 adult plant species were recorded, with 87.2% native and 12.8% exotic species. Some species were exclusively recorded at the upland (29 species) and inundated (27 species) microsites. On average, the species diversity was significantly high (H^I = 3.36). The botanic garden preserves 20 nationally threatened native species, with over half of them listed in the IUCN Red List category of vulnerable species (species facing high risk of extinction in the wild). A considerable number of threatened species were exclusive to the upland (10 species) and inundated (7 species) microsites. The results indicate that the Botanic Garden could play a critical role in biodiversity conservation if integrated into national conservation and urban development strategies to safeguard its potential. The findings of the study are discussed within the context of the contribution of botanic gardens to biodiversity conservation and holds applications for sustainable management of TBGs in urban landscapes.