The aim of this paper is to identify the mechanisms responsible for the chromatic alteration of Pietra Serena sandstone, the stone used in Florentine Renaissance architecture. This stone has a bluish-grey colour when fresh, but is often oxidized acquiring an ochre-brown or reddish colour on buildings. A mineralogical, chemical, petrographic and physical characterization was carried out on the blocks of Pietra Serena used for the Waldensian Church, located in the centre of Florence, showing a characteristic reddish colouring, and on samples from the quarry from which these blocks were extracted. Micro-Raman and SEM-EDS analyses were performed on the red surfaces of the samples in order to highlight the mineralogical and chemical transformations. The results confirmed that the chromatic alteration of Pietra Serena is caused by the mobilization of Fe2+ (prevalent in the clay minerals that make up the matrix of this sandstone and in aluminosilicates such as biotite), which is easily oxidized to Fe3+ in an oxidative environment. This is a consequence of a particular condition of decay, such as the presence of water, CO2, low pH.