Shifts in microbial community structure as a result of land-use, management or climate change could affect soil organic matter dynamics and nitrogen transformations, as these processes may be differently regulated by different soil microbial communities. We conducted a ¹³CO₂ pulse-labeling experiment in differently managed grasslands in Merelbeke (Belgium) to identify the active rhizodeposit-C assimilating microbial communities in different soil habitats, as well as their relative response to several commonly used management practices, including three N-fertilization levels (0, 225 and 450 kg N ha^-1 yr^-1) and two mowing frequencies (3x and 5x yr^-1). Microbial phospholipids fatty acids (PLFA) were extracted from bulk and root-adhering soil samples at 24 h post-labeling in the surface 0-5 cm. Concentrations and ¹³C signatures of selected biomarker PLFAs were determined by gas chromatography-combustion-isotope ratio mass spectrometry. Separate soil samples were incubated for 10 days and potential C and N mineralization was determined. The total PLFA-C concentration increased with 225 kg N additions but decreased at the highest N level. These total PLFA-C changes correlated well with total soil C and N content as well as potential C mineralization. Our ¹³C-PLFA results showed that 5 years of N fertilization negatively affected the relative abundance of the active arbuscular mycorrhizal community while positively affecting that of the gram-positive bacteria. Increased mowing frequency at high N fertilization levels did not affect total microbial PLFA or microbial community structure. A strong negative correlation was found between N mineralization and the ratio of metabolically-active fungal to bacterial PLFA-C but not with total PLFA-C. Our results suggest that microbial community structure, in particular the relative abundance of the active rhizodeposit-C assimilating fungi and bacteria has a large influence on N cycling in these grassland ecosystems.