Free-living nitrogen (N) fixation can be important for sustainable soil fertility, particularly in extensively managed soils with low abundance of leguminous plant species. However, the factors affecting free N-2-fixation in situ are still poorly documented. We investigated the role of microbial active biomass activity, particularly substrate-induced respiration (SIR) and net N mineralization, on the free-living N-2 fixation in soils under a semi-natural grassland ecosystem in France. Analysis of replicated bulk soil and rhizospheric soil samples obtained from sites experiencing contrasting grazing regimes revealed highly significant negative relationships (P < 0.01) between free-living N-2-fixation and SIR or N-mineralization with a significant rhizosphere effect. The study has demonstrated that the activity of free-living N-2-fixers is more important in soils having low active microbial biomass and low N-mineralization rates in these permanent grasslands.