The present work aims to investigate for the first time the materials used in Ribeira Sacra's 16th century murals, as this knowledge would be valuable for research in Technical Art History and for interventions of conservation. A multi-analytical approach is proposed for the investigation of the pictorial palette and technique, and the degradation products. Technical photography (Vis-RAK, IR, UV, micro-photography) and non-invasive analysis by XRF and Vis-RS were performed on site. Micro-samples were taken carefully and studied in the laboratory by means of OM, SEM-EDX, MRS, FTIR, mu-XRD, GC-MS, MALDI-TOF/TOF, and HPLC-ESI-TOF. The analytical results revealed a basic palette of natural red, yellow and brown earth pigments, calcite and several varieties of carbon black. A chromatic enrichment of the palette is observed from the second half of the 16th century, incorporating green, blue and purple colourants. The main pictorial technique is fresco. Some of the pigments, like red lead and azurite, were found altered and the degradation causes and mechanisms were investigated. (c) 2016 Wiley Periodicals, Inc. Col Res Appl, 41, 263-269, 2016