Although amino sugars represent a major component of soil organic nitrogen (ON), the assimilation of nitrate (NO3 −) and ammonium (NH4 +) into amino sugars (AS) by soil bacteria and fungi represents a neglected aspect of the global N cycle. A deeper knowledge of AS responses to N fertiliser addition may help enhance N use efficiency (NUE) within agricultural systems. Our aim was to extend a sensitive compound-specific 15N-stable isotope probing (SIP) approach developed for amino acids (AAs) to investigate the immobilization of inorganic N into a range of amino sugars (muramic acid, glucosamine, galactosamine, mannosamine). Laboratory incubations using 15N-ammonium and 15N-nitrate applied at agriculturally relevant rates (190 and 100 kg N ha−1 for 15NH4 + and 15NO3 −, respectively) were carried out to obtain quantitative measures of N-assimilation into the AS pool of a grassland soil over a 32-d period. Using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) we found that δ15N values for individual AS reflected differences in routing of the applied ammonium and nitrate. The contrasting N-assimilation dynamics of bacterial and fungal communities were demonstrated through determinations of percentage 15N incorporation into diagnostic AS. N-assimilation dynamics of the bacterial community were altered with the applied substrate whilst fungal N-assimilation dynamics were unaffected. Rates and fluxes of the applied N-substrates into the bacterial AS pool reflected known biosynthetic pathways for AS, with fungal glucosamine appearing to be biosynthetically further from the applied substrates than bacterial glucosamine due to different turnover rates. This sensitive and specific compound-specific 15N-SIP approach using AS, building on existing approaches with AAs, enables differentiation of N-assimilation dynamics within the microbial community and assessment of microbial NUE with agriculturally relevant fertilisation rates.