Combined archaeological and ecological investigations in large ancient forests in France have revealed dense networks 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 towards 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 ?15N, indicating the long-term impact of former agricultural practices on forest biogeochemical cycles. 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 millenia of abandonment.