Plant sulphur (S) deficiency is a worldwide concern. However, the mechanisms controlling the immobilization and mineralization of low-molecular weight organic S by soil microorganisms remain unclear. Therefore, we investigated the assimilation of carbon (C) and S by soil microorganisms using uniformly 14C- or 35S-labelled cysteine and methionine. The decomposition of cysteine and methionine in the soil occurred in three steps. First, the microbial biomass (MB) rapidly immobilised the added cysteine-S (55%–63%) and methionine-S (81%–84%) in less than 30 min. Subsequently, S in the MB was released as 35S-sulphate (release of S into the soil peaked at 1 h [21.4%] and 24 h [17.3%] after adding cysteine and methionine, respectively). Lastly, the released 35SO42− was reutilised by microorganisms. The amount of 14CO2 and 35SO42− released from methionine was much lower than that from cysteine. The addition of excess glucose-C or inorganic nitrogen and S had little effect on cysteine and methionine uptake rate, but had a major effect on microbial C use efficiency (CUE) and internal S partitioning and the subsequent release of SO42−. We conclude that the microbial community cycles S-containing amino acids at a high rate, irrespective of soil S and N status with a large proportion of the C used in catabolic processes.