Arbuscular mycorrhizal fungi (AMF) form symbioses with most plants, potentially improving their growth and nutrient assimilation activities. Cysteine (Cys) and methionine (Met) are nitrogen (N)- and sulphur (S)-containing amino acids. Compared with phosphate and N, limited attention has been paid to the role of AMF in low molecular weight organic S acquisition. To explore the uptake and relative contributions of organic and inorganic N and S to plants, and the role of AMF in S uptake, a pot study was conducted based on 14C, 35S, 13C, and 15N quad labelling (6-h labelling test after adding the labelled solution to the soil–plant system) using a mutant tomato genotype with highly decreased AMF symbiosis capacity. Tomato roots can uptake limited amounts of added Met and Cys (<1.77%) within 6 h, as indicated by the 14C and 13C labelling results, and most of them were utilised by soil microorganisms. After uptake for 6 h, 10.0–14.8% of N and 1.4–6.1% of S derived from added Cys and Met were utilised by plants, mainly in inorganic N and S forms derived from Cys and Met decomposition. Met and Cys could be important S sources (Met: 3.0–9.8%, Cys: 8.8–22.0%) for plants, however have negligible roles in N nutrition (∼1%), as most N uptake by plants was derived from soil inorganic N. The tomato uptake of inorganic S derived from Cys decomposition was much higher than that derived from Met, as higher ratios of S-Cys were released as SO42− from microorganisms during the 6-h testing periods. Even with the artificial addition of AMF, most of the added Met and Cys were utilised by Gram-negative bacteria, as indicated by 13C-PLFA biomarkers. AMF reduced host plant uptake of organic N and S, but stimulated plant N uptake from Met and Cys, which was mainly inorganic N following mineralisation. AMF not only utilise organic carbon from host plants but also capture soil organic matter to satisfy their energy demands. In the spaces where both root and AMF occur, AMF colonisation decreased tomato 35S uptake from Cys, Met, and SO42− by 24.6%, 20.6%, and 11.0% within the 6-h uptake period, respectively, when compared with that in the mutant genotype with reduced colonisation capacity; in contrast, AMF colonisation increased 35S-Cys uptake by 118.7% from areas that roots could not reach. Overall, AMF enhanced host plant N uptake but reduced organic N uptake under competition with plant roots for S in the rhizosphere, but stimulated plant S uptake by extraradical mycelia, based on this short-term pulse-chase test.