Protein hotspots in soil, such as those associated with decaying soil fauna or plant litter, may produce ephemeral patches of disproportionately high soil nutrients. These hotspots may occur at the macro- and microscale in close proximity to plant roots, however, the likely concentration of soluble products produced in these hotspots remains poorly understood. To address this, we buried two contrasting biomass residues in soil, namely earthworm (Lumbricus terrestris) and clover (Trifolium repens). Their transformation to amino acids, NH4+ and NO3− were monitored continually over 6 days using microdialysis. All treatments showed greater soluble nitrogen (N) concentrations compared to the unamended controls. The highest concentrations of both amino acids (12.9 mM after 12 h) and NH4+ (45.3 mM after 6 h) were generated in the vicinity of decomposing earthworm. In comparison, dried clover residues yielded 2.7 mM of amino acids at 6 h. After 12 h, amino acid and NH4+ concentrations in both earthworm and dried clover treatments showed a steep decline, returning close to background levels (<20 μM). Through the use of microdialysis we are able to show that soil nutrient hotspots may provide nearby roots with concentrations of amino acids and NH4+ several orders of magnitude higher than found in the bulk soil solution.