Combined archaeological and ecological investigations in a large ancient oak forest in Central France have revealed a dense network of ancient human settlements dating from the Roman period. We demonstrate a strong correlation between present-day forest plant diversity patterns and the location of Roman farm buildings. Plant species richness strongly increases toward the center of the settlements, and the frequency of neutrophilous and nitrogen-demanding species is higher. This pattern is paralleled by an increase in soil pH, available P, and δ15N, indicating the long-term impact of former agricultural practices on forest biogeochemical cycles. These extensive observations in a forested region on acid soils complement and confirm previous results from a single Roman settlement on limestone. Ancient Roman agricultural systems are increasingly being identified in contemporary French forests; the broad extent and long-lasting effects of previous cultivation shown in this study require that land-use history be considered as a primary control over biodiversity variations in many forest landscapes, even after millennia of abandonment.