Global plant sulphur (S) deficiency is increasing because of a reduction in sulphate-based fertiliser application combined with continuous S withdrawal during harvest. Here, we applied 13C, 15N, 14C, and 35S quad labelling of the S-containing amino acids cysteine (Cys) and methionine (Met) to understand S cycling and microbial S transformations in the soil. The soil microorganisms absorbed the applied Cys and Met within minutes and released SO42− within hours. The SO42− was reutilised by the MB within days. The initial microbial utilisation and SO42− release were determined by amino acid structure. Met released 2.5-fold less SO42− than Cys. The microbial biomass retained comparatively more C and S from Met than Cys. The microorganisms decomposed Cys to pyruvate and H2S whereas they converted Met to α-ketobutyrate and S-CH3. The microbial stoichiometries of C, N, and S derived from Cys and Met were balanced after 4 d by Cys-derived SO42− uptake and Met-derived CO2 release. The microbial C:N:S ratio dynamics showed rapid C utilisation and loss, stable N levels, and S accumulation. Thus, short-term organic S utilisation by soil microorganisms is determined by amino acid structure whilst long-term organic S utilisation by soil microorganisms is determined by microbially controlled stoichiometry.