<p>Major advances in metagenomic and biomolecular techniques have opened avenues for the recovery and sequencing of rare and highly fragmented DNA molecules from ancient biological samples, including those of pathogens that may persist in the remains of infected hosts long after their death. Initially consisting of relatively rare findings for a few disease-causing agents of historical importance, the field of ancient pathogen genomics is rapidly progressing towards genome-level analyses of larger sample sets encompassing a wider range of bacterial, viral and eukaryotic taxa. These advances have provided important insights into past pathogen distribution and genomic make-up, and the unprecedented opportunity to track their evolution ‘in action’ over large timescales. In this Review, we explore the major contributions of ancient DNA research to the understanding of pathogen evolution and its association with past epidemics, as well as human sociocultural and migration history, including numerous important studies that have been published in recent years. Moreover, we discuss existing limitations and future prospects of ancient pathogen genomics and the relevance of the field to current public health challenges.</p>