Sixteenth-century mural paintings of Nossa Senhora das Neves in Vilar de Perdizes (Galicia—North Portugal Euroregion) were analysed. An iconographic study has allowed us to understand the meaning of the seven scenes that constitute the mural painting. X-ray fluorescence (XRF) and Raman spectroscopies determined the compounds used in this mural painting, both in the original and in later repaintings. The black paint was bone black. Hydroxyapatite characteristic bands and those of the associated phosphates have been identified. White lime was used as white paint. Lepidocrocite and goethite were used to make yellows, and hematite was used to make red shades. Cinnabar has been used for a later red repaint. Carbon-based compounds and rutile were used to create different tonalities by darkening or lightening colours. It is of great significance to obtain accurate and reliable mural painting information through scientific means, since preservation, restoration, and repainting without detailed information can be harmful to mural paintings.