This paper is based on a laboratory study of 15th-century paintings attributed to Nuno Gonçalves, namely the Polyptych of St. Vincent, the Martyrdom of St. Vincent and Four Saints sets. According to results of optical microscopy, energy dispersive X-ray fluorescence spectrometry and micro-X-ray diffractometry analysis, the preparation layers are made of “gesso grosso”, mostly anhydrite and, in lower quantities, gypsum. Aluminosilicate, quartz, dolomite, celestite, and phosphorous and led compounds were also minor constituents in these layers. Regardless of overall similarities, differences were found between the Polyptych and the other sets in terms of its constituents. The study also found that the preparation of each painting has very uneven thickness, sometimes far superior to the values determined in previous studies. By the use of infrared microspectroscopy, a proteinaceous material and oil were found. This, however, may not have been used in the preparation, but result from contamination from the upper layers.