The painting depicting the “Virgin with the Child and two angels” by a Tuscan anonymous artist of the XIII century, has raised great interest regarding the execution technique including the use of silver, applied on wood as a background. An in-depth investigation was carried out especially concerning the burnishing process of the silver leaves as well as a detailed inspection of the nature of the organic compounds over the metal leaf and the degradation products of the silver layer. To this aim, a multianalytical approach was used including UV–VIS microscopy along with an Ultra-High-Resolution Scanning Electron Microscopy (UHR-SEM), as well as Fourier Transform Infrared (FTIR), fluorescence and micro-Raman spectroscopies. As a result, the presence of an oil-resin varnish layer in the uppermost layer and a wax-protein layer between the varnish and the metal layer as well as the evidence that the silver leaf was applied with high accuracy directly to the preparation layer, were clearly demonstrated. Furthermore, degradation products of the silver leaf were identified as sulphides, chlorides, and oxides. The holistic approach herein adopted enabled a step forward in the knowledge of the 13th century silver leaf gilding technique, adding value to the originality of this artwork. In this respect, the scientific evidence led to the assumption that likely this painting originally showed a silver-colored background (“argento biancheggiato”) as compared to the initial theory of a case of “argento deaurato”.