Riverine habitats play a vital ecological role, offering key ecosystem services, regulating hydrology, and supporting high biodiversity, including species and habitats of conservation concern. This study aimed to identify Annex I habitats under the Habitats Directive (92/43/EEC) within a former protected area along the mid-upper Elsa River (Tuscany, central Italy). From 2021 to 2023, 85 vegetation relevés were collected to classify habitats, map their distribution, and assess major threats. Multivariate analyses revealed 15 Annex I habitats, including 32A0, reported here for the first time in Italy, and three priority habitats (7220*, 91E0*, 91AA*). Habitat 32A0, officially included in Annex I following the accession of Croatia to the EU, shows distinct ecological and structural features compared to habitat 7220*, despite sharing some bryophyte species. While the two often occur in close proximity or form mosaics, habitat 32A0 develops in active waterfalls with constant flow and low carbonate deposition, whereas habitat 7220* forms in slow-dripping areas with high tufa accumulation. The absence of habitat 32A0 from the Italian Habitat Manual underscores the need to revise national habitat classifications to improve identification accuracy and conservation strategies. Our study enhances knowledge of riverine habitats and stresses the importance of adaptive management to safeguard the Elsa River ecosystem. Key actions include continuous monitoring and control of invasive alien species. Incorporating the area into a nearby Special Area of Conservation would strengthen long-term protection in line with the EU Biodiversity Strategy for 2030.