Serpentinite habitats represent unique ecosystems that support specialized plant communities adapted to nutrient-poor, metal-rich substrates. The Brdjani Gorge in central Serbia is one of the country's most important serpentinite localities; however, its ecological integrity is increasingly threatened by anthropogenic pressures, particularly the proximity of a municipal landfill and transport infrastructure. The aim of this study was to provide a comprehensive assessment of the floristic diversity of the Brdjani Gorge, evaluate the conservation importance of its flora, and identify priorities for the protection of habitat-specific plant species. Field investigations were carried out in 2023 and 2026 within the Rufford Foundation-funded projects. The research was conducted in a selected section of the Brdjani Gorge harboring ecologically significant plant species. Plant taxa were recorded, photographed, collected, and identified using standard floristic literature and current taxonomic databases, and the flora was further analyzed according to life-form and chorological spectra. The survey documented 119 vascular plant species belonging to 85 genera and 36 families. The flora includes several taxa of high conservation significance, including the endemic Halacsya sendtneri, the relict fern Hemionitis marantae, and serpentine-associated species such as Asplenium adulterinum and Silene paradoxa. The coexistence of endemic, relict, obligate, and facultative serpentinophytes reflects the exceptional ecological value and evolutionary importance of this ultramafic habitat. Despite its remarkable biodiversity, the Brdjani Gorge is exposed to habitat degradation caused by human activities, which may threaten the persistence of its specialized flora. The findings emphasize the need for continuous biodiversity monitoring, effective habitat management, and stronger conservation measures to preserve one of Serbia's most valuable serpentinite ecosystems. Protecting this locality is essential for maintaining regional plant diversity and safeguarding rare species associated with ultramafic environments.