The authors also wish to acknowledge to the HERCULES Lab and José de Figueiredo Lab teams for all scientific collaboration, in particular, Luis Piorro for performing the IRR exams, Maria José Oliveira for XRD analysis and Claudia Pereira at Museums and Conservation library. The authors also wish to thank the National Ancient Art Museum (Lisbon) for granting access to the painting and to Centro de Estudos Históricos da Lourinhã. Publisher Copyright: © 2019 by the authors. Licensee MDPI, Basel, Switzerland. ; The painting collection of Santa Casa da Misericórdia da Lourinhã is amongst Portugal’s most notable and scarcely best-known cultural heritage. The artistic interest of this pictorial group, besides the advanced state of degradation of a number of the paintings, together with the ruined circumstances of the building accommodating the collection, today in reconstruction, were the key reasons for this study. Thermo-hygrometric measurements were carried out. A multianalytical methodology incorporating micro-X-ray diffraction (µ-XRD), energy-dispersive X-ray fluorescence spectroscopy (EDXRF), scanning electron microscopy–energy dispersive spectroscopy (SEM–EDS), micro-Raman spectroscopy (µ-Raman), micro-Fourier transform infrared spectroscopy (µ-FTIR) has been followed for the study. These analyses were complemented by infrared photography (IRP) and reflectography (IRR), allowing the study of the underdrawing technique. The results of this study were compared with previous ones of the painter’s workshop and important distinctions and similarities were found within the materials and techniques used. This analysis methodology on materials contributes to safeguarding and the ensuing community awareness of this cultural heritage in danger. ; publishersversion ; published