Research on the materiality of Andean Colonial artistic heritage contributes to the knowledge on the cultural and social history of the region. In this work we present the results of an interdisciplinary and multi-analytical investigation on a mural painting of the church of Curahuara de Carangas in Bolivia. This painting has been hidden for years behind the main 18th century wooden altarpiece. Its stylistic and pictorial characteristics place it in the early 17th century (1608), while other mural paintings of the temple correspond to a second stage, by 1777. Pigments were identified by scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS) and Raman micro-spectroscopy. The application of imaging methods not only allowed the identification and characterization of the materials but also gave information on the manufacturing technique of this mural painting. An outstanding finding in our work was the identification of a mixture of two copper minerals, atacamite [Cu2Cl(OH)3] and antlerite [Cu3SO4(OH)4], as the green pigment. Atacamite and antlerite have been separately identified in a polychrome sculpture and a mural painting, respectively, but this is the first report on their use as a mixture. Other pigments identified in the mural painting were haematite, azurite, red lead, cinnabar, gypsum, cerussite, orpiment, and carmine lake. These results, together with the identification of traces of minerals of the region, indicate the use of local materials by Andean painters from the early Colonial period. This suggests continuity in the cultural practices from pre-Hispanic times and an appropriation and reformulation of traditional European painting practices adapted to new resources. Analysis of lipid and protein materials by gas-chromatography coupled to mass spectrometry (GC–MS) indicated the use of a secco painting technique involving the use of egg as pigment binder. Identification of animal glue suggested its use as the plaster primer. The knowledge of the materials and the painting technique of this artwork will allow the identification of similarities and differences with other mural paintings in churches of the Andean region and give useful information for conservation and preservation of this artwork.