Sulphur-containing amino acids (i.e. Cysteine (Cys) and methionine (Met)) constitute an important proportion of the soil organic sulphur. However, detailed information regarding the microbial transformation of Cys and Met at a molecular level remain poorly characterized. To trace the fate of carbon (C) and sulphur (S) derived from Cys and Met in an agricultural grassland soil, a14C and 35S dual-isotopic labelling approach was adopted. We also investigated whether their mineralization was affected by manipulating C (added as glucose), nitrogen (N), phosphorus (P) and S (added as NH4NO3, KH2PO4 and K2SO4) availability in soil solution. Our results showed that over a 7-day incubation period, 67.2–89.2% of the 14C derived from Cys and Met was respired as 14CO2, 2.7–19.5% had been immobilized in the soil microbial biomass; while the recovery of 35S in soil solution ranged from 6.4 to 9.9%, with the reminder retained in the soil microbial biomass. Overall, our results indicated that soil microbial communities possess a high capacity to utilize Cys and Met. Furthermore, using the 14C and 35S dual-labelling technique, we found that C and S derived from Cys and Met were microbially mineralized and immobilized at different rates, indicating that the cycles of these two elements were temporally decoupled at the molecular level. The addition of glucose-C increased 14CO2 respiration from Cys and Met after 7 d, while in comparison inorganic N, P and S addition had less effect on 14C and 35S partitioning.