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Human microbiome studies are increasingly incorporating macroecological approaches, such as community assembly, network analysis and functional redundancy to more fully characterize the microbiome. Such analyses have not been applied to ancient human microbiomes, preventing insights into human microbiome evolution. We address this issue by analysing published ancient microbiome datasets: coprolites from Rio Zape ( n = 7; 700 CE Mexico) and historic dental calculus ( n = 44; 1770–1855 CE, UK), as well as two novel dental calculus datasets: Maya ( n = 7; 170 BCE-885 CE, Belize) and Nuragic Sardinians ( n = 11; 1400–850 BCE, Italy). Periodontitis-associated bacteria ( Treponema denticola , Fusobacterium nucleatum and Eubacterium saphenum ) were identified as keystone taxa in the dental calculus datasets. Coprolite keystone taxa included known short-chain fatty acid producers ( Eubacterium biforme, Phascolarctobacterium succinatutens ) and potentially disease-associated bacteria ( Escherichia , Brachyspira) . Overlap in ecological profiles between ancient and modern microbiomes was indicated by similarity in functional response diversity profiles between contemporary hunter–gatherers and ancient coprolites, as well as parallels between ancient Maya, historic UK, and modern Spanish dental calculus; however, the ancient Nuragic dental calculus shows a distinct ecological structure. We detected key ecological signatures from ancient microbiome data, paving the way to expand understanding of human microbiome evolution. This article is part of the theme issue ‘Insights into health and disease from ancient biomolecules’.