Sulfur vacant sites in MoS2 nanosheets (NSs) have been confirmed to be effective in reacting with thiol molecules in solution. However, little-to-no information is known about the molar ratio of the remaining thiols and the produced disulfides in the reaction of MoS2 NSs and thiol molecules. Additionally, the MoSe2 NS-mediated conversion of thiol molecules has not been explored at present. Herein, we conducted a comparative study regarding the very-few-layer (VFL; layer number ∼ 3) MoS2 and VFL-MoSe2 NS-induced conversion of glutathione (GSH) to GSH disulfide (GSSG) in phosphate-buffered saline and the oxidation of GSH in live cells. Compared to traditional colorimetric assays for determining the percentage loss of GSH, liquid chromatography and mass spectroscopy were introduced to confirm and accurately quantify the molar ratio of the remaining GSH to produced GSSG from the reaction of the present VFL-MoS2 and VFL-MoSe2 NSs with GSH at pH 4.0–10 and 25–70 °C. The produced GSSG molecules were the leading product in the above-discussed reaction, and most of them were liberated into the solution rather than adsorbed on the NS surface. Additionally, it was found that VFL-MoSe2 converted more efficiently GSH to GSSG than VFL-MoS2 NSs. These findings were also valid for 5-thio-2-nitrobenzoic acid, which was well-suited to evaluate the difference in the rate constant of thiol conversion among the proposed two-dimensional NSs. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assay, GSH-specific fluorescent probe, and live/dead cell staining kit demonstrated that the cellular cytotoxicity of the proposed two-dimensional NSs was highly correlated with the oxidation of intracellular GSH. The presence of selenocysteine filling selenium vacancies in VFL-MoSe2 NSs suppressed the conversion of GSH to GSSG in solution and the oxidation of GSH in live cells. Our findings point out that the reaction pathway of MoS2 and MoSe2 NSs with thiols not only involves vacant site repairing and thiol conjugation but also includes thiol dimerization.