The study of ancient gilding is often problematic, as the gilding layers are soft and prone to deformation during sample preparation. In this respect, focused ion beam (FIB) milling on a field emission gun scanning electron microscope (FEG-SEM) provides poorly invasive in situ sampling. The operating process is here detailed and applied on gilded medieval copper-based elite jewellery (10th century) from Prague Castle. Obtained cross-sections and slices of gilded samples were investigated up to nanometer scale without gilding layer deformation. By coupling structural observation with elemental X-ray analysis (EDS) and electron beam diffraction (EBSD), FIB FEG-SEM provided new data on the physical-chemical characteristics of the gilded layer. The gilding has a two-layer metallurgical structure containing a quaternary Au (Hg, Cu, Ag) alloy corresponding to the gold solid solution fcc phase, and a submicrometric inner sublayer formed by a Au-Cu phase, relative to the inter-diffusion of copper from the substrate during the fire gilding process. The applied temperature for gilding can be estimated at c. 400 • C. Sintering of the mercury-gold amalgam globules during the fire heating is highlighted. The precise characterisation of the gilding layers provided useful comparative parameters for identifying fire gilding skill levels and assessing the overall quality of the archaeological pieces.