The 16th century Florentine Codex, realized by Mesoamerican artists under the guidance of the Spanish Franciscan Bernardino de Sahagùn, is a milestone in the anthropological studies by European scholars to define the pre-Columbian Mesoamerican past, and depicts the New World as it was caught in the crucial moment of the early conquest. This chapter reports on a representative selection of colours (and drawings), among 150 investigated areas of the manuscript, to support the theory that the choice of colours obey precise and significant symbolic values, which highlights the will of the author, fray de Sahagún, to transmit precious information to readers for understanding the pre-Columbian culture. The reported images were selected following criteria of historical and symbolic meaningfulness, while trying to cover an even distribution between the different books and figures, so to analyse a variety of painting techniques, and a full palette of colours. The results were thus compared with the information on colours and on the traditional manufacturing procedures, reported in some of the Codex chapters, and with laboratory model samples prepared accordingly. The analysis unveiled the criteria behind the usage of organic and inorganic colours for depicting different topics. Moreover, it highlighted the use of mixed Mesoamerican and European painting techniques, emphasizing the cultural fusion that found in the Florentine Codex one of its first expressions.