Microbial contribution to carbon and nitrogen cycling in forest soils is important, and may depend on tree species. The amount of amino sugars and their ratios can serve as reliable indicators for bacterial and fungal contribution. We compare forest floor microbial residues (amino sugars) beneath three canopy-tree species (Sugar Maple (SM), Acer saccharum Marsh; Basswood (BA), Tilia americana L.; Eastern Hemlock, Tsuga canadensis L.) replicated in five plots in an upper Michigan old-growth forest. We hypothesized that because individual tree species develop a unique microbial community over time, they will accumulate microbial residues to different degrees. In this study at three tree species sites, the absolute amount of fungal residue (glucosamine (GluN)) was relatively constant, while absolute quantities of bacterial residues (galactosamine (GalN) and muramic acid (MurA)) were least in the Hemlock site. Amino sugar ratios revealed that microbial residues were compositionally distinct in the three sites. The lower ratios of GluN to GalN and GluN to MurA in BA and SM sites relative to Hemlock site indicate the lower net accumulations of GalN and MurA in Hemlock site. In terms of microbial contribution to carbon and nitrogen cycle in forest soils, we suggest that caution may be needed when using amino sugars as a tool, especially for nitrogen pool assessment, as the amino sugars are diluted by plant-derived litter. This study provides information on the microbial residues in undisturbed forest soils which may assist interpretation of data derived from managed or damaged forests in the future.