This article describes a study carried out on a mural painting in the Convent of Santo António dos Capuchos in the town of Estremoz (southern Portugal). Experimental work was undertaken to identify pigments and to elicit hypothesis about their local origin. Another aim was to ascertain the painting technique (binders, stratigraphy) and to try to investigate the causes that led to the deterioration of the blue and green pigments. Elemental and mineralogical analyses of the pigments were performed by x-ray diffraction (μ-XRD), portable energy dispersive x-ray fluorescence (EDXRF) and scanning electron microscopy (SEM), complemented by microchemical tests and Fourier transform infrared spectroscopy (FTIR). These techniques have shown that a variety of pigments were used, namely earth pigments (red and yellow ochres, green earth and probably black earth and umber), copper pigments (azurite, malachite), probably from the Estremoz region, and smalt, used pure or in mixtures. The results also revealed the presence of salt veils, biological colonization and possibly secco applications with lime (lime painting), which may have contributed to the actual state of conservation of fresco.