Diagnosis of ancient infectious diseases has seen remarkable advances, contributing significantly to understanding historical health challenges and shaping medical research. This review explores the evolution of diagnostic methods and features emerging diagnostic techniques. The early detection of ancient infectious diseases primarily relied on observational evidence, skeletal remains, and historical records. Notable outbreaks such as the Black Death in the 14 th century and smallpox epidemics, have left historical imprints. Pioneering diagnostic methods included the paleopathological analysis of the remains of bones and tissue lesions.

Later, microbiology methods were adapted to ancient materials, including molecular and serological techniques such as PCR, serology and immunodetection, which were employed to detect DNA or antibodies from ancient pathogens under the name of the paleomicrobiology approach. This emerged as a distinct field, making it possible to isolate and identify ancient pathogens from preserved samples. Advances in next generation sequencing are now revolutionising the diagnosis of ancient diseases, enabling the retrieval of ancient pathogen genomes from well-preserved samples such as dental pulp, tartar, bone and mummified tissues, helping understand disease evolution and transmission patterns. Ongoing improvements in DNA sequencing and metagenomics will enhance the accuracy and scope of the diagnosis of ancient diseases. Collaboration between archaeologists, historians, paleomicrobiologists, geneticists and epidemiologists will drive further breakthroughs. Advances in paleocytology, metagenomic analysis, including non-invasive techniques, such as mass spectrometry, may offer new avenues for identifying ancient pathogens. Current technologies and interdisciplinary collaboration hold promise for uncovering even more insights from the past and potentially informing future disease prevention strategies.