Human activities have affected the surrounding natural ecosystems, including belowground microorganisms, for millennia. Their short- and medium-term effects on the diversity and the composition of soil microbial communities are well-documented, but their lasting effects remain unknown. When unoccupied for centuries, archaeological sites are appropriate for studying the long-term effects of past human occupancy on natural ecosystems, including the soil compartment. In this work, the soil chemical and bacterial compositions were compared between the Roman fort of Hegra (Saudi Arabia) abandoned for 1500¿years, and a preserved area located at 120¿m of the southern wall of the Roman fort where no human occupancy was detected. We show that the four centuries of human occupancy have deeply and lastingly modified both the soil chemical and bacterial compositions inside the Roman fort. We also highlight different bacterial putative functions between the two areas, notably associated with human occupancy. Finally, this work shows that the use of soils from archaeological sites causes little disruption and can bring relevant information, at a large scale, during the initial surveys of archaeological sites.