The artist Johannes Aquila was active in the last quarter of the fourteenth century in the area of Middle East Europe, divided today between Slovenia, Austria, and Hungary. He is principally known for his mural paintings in Velemér (Hungary), Turnišče and Martjanci (Slovenia), Bad Radkersburg and Fürstenfeld (Austria). His works have been studied from the art-historical point of view, revealing a complex influence on his style, from the Italian Trecento and from the Northern, especially Czech art. The present research aims to understand whether materials and painting techniques used by Aquila and his workshop also correspond to the North European or rather South European, mainly Italian, contemporary painting manner. Different invasive and non-invasive techniques have been selected in all five locations, depending on permissions obtained and analytical equipment available. Besides a precise study in situ by the naked eye and by digital microscope, also a portable X-ray fluorescence (XRF) and VIS spectrophotometry were used. Where possible, microsamples of pigments and plasters were extracted and studied by optical microscopy, SEM–EDX, FTIR, XRF and XRD. The results showed that artists used mostly inorganic pigments, generally stable in a fresco technique, and probably some lead-based ones. Aquila and his disciples combined a fresco, a secco and sometimes lime technique. His plasters are poor in lime and can contain organic fibres. There is a huge difference in the quality of technical execution between his early murals and his latter ones, where most of the work was carried out by his workshop.