The application of paleoimmunological techniques on ancient human bone tissue allowed the identification of ancient pathogens, such as malaria by Plasmodium falciparum. Until now the immunocromatographic tests were applied only on mummified muscle. Ir this study positive results were obtained using ancient spongy bone samples belonging to the skeletal remains of four members of the Medici family of Florence who, according to archive data, died of malaria: Eleonora di Toledo (1522-1562), Giovanni (1543-1562) and don Garzia (1547-1562), and the Grand Duke Francesco I (1531-1587). These results are of particular interest not only because they confirm the historical data, but also because they demonstrate that immunological tests can be applied to bone samples, opening new paths of research for the screening of large skeletal series.