Dissolved organic nitrogen (DON) represents an important soluble nutrient pool in soil, however, little is known about the dynamics of DON in the litter and topsoil of Brazilian sugarcane (Saccharum spp.) fields, particularly those that are harvested mechanically, without burning. Therefore, the aim of this study was to determine the microbial mineralisation and sorption affinity of DON compounds in litter and soil from the litter-soil transition zone of two sugarcane plantations located in southeastern Brazil. We directly measured the C mineralisation of 14C-labelled amino acids (mix of 17 amino acids), peptides (L-dialanine and L-trialanine), urea, and protein (isolated from tobacco leaves) by capturing 14CO2 evolved from the litter and soil over 168 h. A sorption assay was performed using the same treatments. We found differences in the organic and mineral N pools of the litter and soil, as well as in microbial community composition. Except for protein in the soil, the DON compounds were taken up rapidly by microbes. However, the C use efficiency was higher for the amino acid mix than for the peptides and urea, indicating more rapid post-uptake catabolism (with subsequent mineralisation as 14CO2) of both compounds. In addition, protein had the highest sorption affinity, especially in soil, and the weak sorption affinity of the amino acids, peptides, and urea indicates moderate bioavailability of these fractions to microbes and plants. We conclude that strong sorption of protein to the solid phase limits its bioavailability and represents a rate limiting step in DON turnover.