Ancient Rome was the capital of an empire of ~70 million inhabitants, but little is known about the genetics of ancient Romans. Here we present 127 genomes from 29 archaeological sites in and around Rome, spanning the past 12,000 years. We observe two major prehistoric ancestry transitions: one with the introduction of farming and another prior to the Iron Age. By the founding of Rome, the genetic composition of the region approximated that of modern Mediterranean populations. During the Imperial period, Rome’s population received net immigration from the Near East, followed by an increase in genetic contributions from Europe. These ancestry shifts mirrored the geopolitical affiliations of Rome and were accompanied by marked interindividual diversity, reflecting gene flow from across the Mediterranean, Europe, and North Africa.

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Like other hypogeal environments, the Roman Houses of the Caelian Hill are prone to unwanted biological growth. Wide conservative interventions have been carried out at the beginning of this millenium to reduce biodeterioration and physical–chemical damages. Retracing the last monitoring work, we assessed the site’s current state of conservation and biodeterioration intending to check the previous treatments’ effectiveness and deepen the common knowledge of the subterranean biota and their possible biodeteriogenic effects. Starting from the past test areas and the previous identifications of the occurring biodeteriogens, we further isolated and identified the main eubacterial, fungal, and phototrophic settlers, focusing on some detrimental traits for wall paintings (i.e., acid production and carbonate precipitation). The achieved results proved the success of the performed interventions in reducing the wall’s water content. Otherwise, the new conditions raise, in the long term, new concerns about lampenflora, carbonate precipitations, and salt efflorescence. Here, the Caelian Houses’ new status is documented. The possible favouring conditions for the different groups of biodeteriogens, along with the taxonomical novelties, additional risks tied to the anthropization of the resident culturable microbial community, and the possible relation between the black fungus Cyphellophora olivacea and roots, are reported and discussed.