The Madonna with Child by Andrea Mantegna owned by the Museo Poldi Pezzoli in Milan is painted on canvas with an unusual distemper technique. During the period of 1863–1865, the painting was restored by Giuseppe Molteni. The identification of potential retouchings by Molteni, possibly covering part of the original layer, was the object of this work carried at the Opificio delle Pietre Dure. To evaluate the extent of both Molteni’s intervention and Mantegna’s original layer, the MA-XRF spectrometer developed by CHNet-INFN was used to discriminate between the two paint layers and identify the materials and the extension of both “artists”. Indeed, the elemental maps showed that Molteni’s work entirely covered the mantle of the Virgin, even changing the fold of the draperies and enriching the red robe with shell gold highlights, giving a different appearance to the painting. Moreover, MA-XRF also revealed that the original Mantegna was still mostly intact underneath Molteni’s layer, thereby providing a decisive guide for conservation works. These results indeed formed the basis for the technical decision to remove the varnish and Molteni’s version, unveiling the original Mantegna. A second MA-XRF campaign was then carried out to fully characterise the materials of this unusual painting technique.