Several pre-Columbian mummies are preserved in the Museum of Anthropology of the University of Florence. These bodies were brought from South America to Italy in the second half of the 19th century. We studied the natural mummies of two adults dated back between 1410 and 1530. Complete autopsies, histological and genetic analyses were performed. Histopathological study on the tissue specimens of the lungs of the first mummy showed numerous alveoli, in large fibrous stroma, areas of pulmonary anthracosis and a massive presence of alveolar exudate compatible with the diagnosis of bronchopneumonia. The other corpse presented a marked megavisceral syndrome characterized by megacolon, megaoesophagus and cardiomegaly. Amastigotes of Trypanosoma cruzi were clearly detected in the myocardial fibers. DNA was extracted and the shotgun metagenomic sequencing showed bacterial and fungal taxa that may resemble both the thanatomicrobiome and extant human gut microbiomes. Bacterial groups present in the mummies included the Bacillales, Enterobacteriales and, especially, the Clostridiales; it is feasible to hypothesize that these individuals were exposed to these pathogens by the ingestion of contaminated food and water. Furthermore, we detected the presence of sequences of Trypanosoma cruzi, Leishmania donovani and antibiotic-resistance genes. The presence of antibiotic-resistance genes clearly indicates that these genes pre-date therapeutic use of these compounds and that they are not necessarily associated to a selective pressure of antibiotics use. Studies on the ancient microbiome represent an opportunity to better understand microbe-host interactions, the membership and ecology of microbes, the evolution of commensal and pathogenic microorganisms and their impact in health and disease.