Amino sugars (AS) are the building blocks of microbial cell walls and can be stabilized in the soil after cell death. Their total concentration in the soil has been used as an indicator of microbial contributions to soil organic matter (SOM). Since bacteria and fungi have different cell wall structures, relative abundance of their characteristic AS has been used to assess fungal and bacterial contributions to SOM. However, little is known about the dynamics of fungal and bacterial-derived AS and their role in soil carbon sequestration. In our study, compound-specific 13C analysis of AS via gas chromatography combustion isotope ratio mass spectroscopy was used to investigate the impact of substrate quality on the dynamics of fungal and bacterial AS in a sandy soil under laboratory conditions. Soil samples were incubated for 184 days with five different substrates (wheat roots, leaves, hot-water extracted leaves, stems, grain) enriched in 13C and differing in C/N, lignine/N, hemicellulose and cellulose content. During the entire incubation, respiration was measured and substrate- and native SOM-derived respiration as well as the priming of native SOM by substrate addition were compared across the five treatments. Substrate-derived AS-C concentrations were determined after 1, 2, 4, 8, 14, and 18 weeks in the grain, leaves and root treatment. We observed significant negative correlations between respired C and both lignine/N and hemicellulose, and between primed C and cellulose. Substrate-derived total AS-C concentrations were highest in the high-quality grain, and lowest in the low-quality root treatment. In all treatments, greater bacterial- than fungal-derived total AS-C was found. While this trend was also observed for substrate-derived AS-C in the root treatment, the opposite was found in the grain treatment. A significant negative correlation between C/N, lignin/N as well as hemicellulose and substrate-derived fungal AS-C suggested stimulated fungal pathways with higher quality substrate.