Electron backscatter diffraction (EBSD) was used in Conservation Science for characterization of ancient materials collected from works of art. The results demonstrate the feasibility of EBSD analysis on heterogeneous matrices as very small samples of paint layers collected from paintings. Two reference pigments were selected from those used by artists to investigate the relationship existing between EBSD pattern quality and properties of the investigated material (i.e., average atomic number, density, and Mohs hardness). The technique was also tested to investigate the pigment phases on two real samples collected from Romanino's Santa Giustina altarpiece, an oil on wood painting dated 1514 (Civic Museum, Padova, Italy). Results show for the first time the acquisition of EBSD patterns from painting samples mounted in resin, i.e., painting cross sections, opening a new powerful tool to elucidate the pigment phases avoiding large sampling on works of arts and to further study the complex mechanisms of pigment deterioration.