Eastern Orthodox religious panel paintings (“icons”) are continuously manufactured since the dawn of Christianity and are still being used in the framework of pertinent rituals and worship. In the current work, an important Greek icon is investigated through non-invasive macroscopic X-ray fluorescence (MA-XRF) analysis to reveal the employed painting materials and techniques. The artifact in consideration was painted by the renowned icon painter Theodoros Poulakis who was active during the 17th century (ca. 1622–1692). Poulakis’ work is characterized by the extremely skillful painting and played a major role in the stylistic developments of Greek religious painting; additionally, more than one hundred icons that either bear Poulakis’ signature or are attributed to his hand on stylistic basis are spotted today. Through the MA-XRF investigation of the icon in consideration authors were able to identify the materials employed in its manufacturing and revealed interesting technical details, including some peculiar –and possibly idiomorphic– characteristics. In particular, the identified pigments include vermilion, lead white, minium, at least two distinct ochre pigments, a copper-based blue (presumably azurite) and two distinct (in terms of composition) copper-based green pigments, along with a deep-red organic lake. Poulakis used extensively high-purity gold leaves for gilding the background and the haloes of the figures, while MA-XRF allowed for the identification of inferior (in terms of purity) gold leaves that were used for rendering gilded highlights on various details. The analytical data allowed for an in-depth investigation of the materials and techniques employed by Poulakis in this particular painting and led to the spotting of some rather idiomorphic technical characteristics, such as the presence of a copper green pigment that contains Zn–As–Ba impurities and the combination of multiple colors for highlighting red vestments. The results are evaluated under the light of other relevant studies and aim primarily at setting a sound foundation for a forthcoming, extensive and thorough investigation of the materials and techniques employed in other Theodoros Poulakis paintings.