The aim of this work was to investigate differences in soil chemistry and understory composition between recent forests (sites afforested in the last 170 years) and ancient forests growing on non-acidic soils. The study was carried out on hardwood forests at moderate elevation (400–600 m asl) in the Jura Mountains (N.E. France) on four main pedological substrates with different characteristics. The floristic composition of 127 stands from recent forests (n = 65) or ancient forests (n = 62) was surveyed. Some functional traits and the Ellenberg indicator values of the surveyed species were recorded. In addition, the topsoil from 30 stands was analysed. The composition of the flora was analysed by Detrended Correspondence Analysis and the species which were typical of one class of forest age were identified using a chi-square (x2) test. The difference between forest classes for plant traits, their indicator values, or soil chemistry was tested using the generalized linear model and Bonferroni t-tests (or Kruskall– Wallis tests). The floristic composition of the ancient forests was significantly different from that of the recent forests and was characterized by a high occurrence of shrub species in recent forests. These differences were associated with higher specific leaf area, low-range seeds dispersal, and some life forms like geophytes. There was no clear difference in soil chemistry between the two classes of forests, except for d15N values. The weakness of the difference in the soil between ancient and recent forests suggested that changes in soil chemistry caused by a former agricultural land use were not responsible for the differences in understory composition recorded. The differences in functional traits between the two forest classes supported this conclusion. We finally concluded that (i) past land use modifies the vegetation composition of current forests, even on neutral soils and that (ii) in our context, biological filters were probably responsible for these changes.