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The influence of ageing grassland on microbial community structure in different long-term grassland regimes compared to tillage in neighbouring fields was investigated to evaluate whether grassland restoration can be considered as a specific type of management for soil conservation in northern France. Microbial community structure was examined by analyzing the distribution of total and labile organic matter, the size of bacterial and fungal populations, and bacterial metabolic fingerprints and fungal genetic fingerprints. Results showed a gradual positive increase of total microbial biomass between the intensive management reference site and the six grassland soils, and that soil organic matter storage is associated with changes in microbial biomass. There was a large increase in fungal and bacterial populations in the permanent grassland, but bacteria were more weakly affected by agricultural management practices than the fungi. Although potential functional diversity shifts in the bacterial community seemed to be related to the ageing grassland gradient, we were not able to highlight any significant difference in bacterial genetic diversity between the sites. There was, however, a strong relationship between fungal genetic diversity and the ageing grassland. Finally, an increase in microbial activities (% mineralization) was observed according to the age of the meadow. Among agricultural management practices, grassland restoration may have a positive impact in maintaining the soil status.