Nitrogen (N) and sulphur (S) are essential nutrients for plant growth. A pot experiment was conducted to verify whether maize and soybean under monoculture and intercropping could utilise N- and S-containing amino acids when competing with soil microorganisms. Maize and soybean were able to utilise methionine (Met; 1.9–2.2% of total addition) and cysteine (Cys; 0.6–1.6% of total addition) as N and S sources, however they faced competition with soil microorganisms. Six hours after microbial uptake, 45.3–64.0% of the S from Met was retained in the microbial biomass, while there was much lower retention from Cys (16.8–34.5%), and 32.5–44.1% of the S from Cys and 15.6–33.3% of that from Met was transformed to SO42−. Cys was a superior S source for plants compared to Met, as higher SO42− release from Cys could support plant growth. Both maize and soybean plants took up NH4+ (98–99% of total N uptake) and SO42− (85–90%) as their main N and S sources from soil. The N from Cys and Met accounted for only ~1% of total N uptake from soil, and organic Cys and Met accounted for only ~0.2% of the total N uptake, indicating that these two amino acids have limited effects on plant N nutrition, due to the high inorganic N content in agricultural soil. However, the Cys and Met contribution (i.e., organic S uptake and mineral S uptake originating from Cys and Met) to total S uptake (10–15%) was an order of magnitude higher than their N contributions, suggesting that Cys and Met play an important role in soil S nutrition. Intercropping altered the uptake but not the preference for N and S forms in maize and soybean. Overall, the results suggest that S-containing amino acids are important S sources for plant growth even at a lower concentration in soil, but that they play a limited role in plant N nutrition due to a larger inorganic N pool in agricultural soil.