TFM (L-trifluoromethionine), a potential prodrug, was reported to be toxic towards human pathogens that express MGL (L-methionine γ-lyase; EC 4.4.1.11), a pyridoxal phosphate-containing enzyme that converts L-methionine into α-oxobutyrate, ammonia and methyl mercaptan. It has been hypothesized that the extremely reactive thiocarbonyl difluoride is produced when the enzyme acts upon TFM, resulting in cellular toxicity. The potential application of the fluorinated thiomethyl group in other areas of biochemistry and medicinal chemistry requires additional studies. Therefore a detailed investigation of the theoretical and experimental chemistry and biochemistry of these fluorinated groups (CF₃S⁻ and CF₂HS⁻) has been undertaken to trap and identify chemical intermediates produced by enzyme processing of molecules containing these fluorinated moieties. TvMGL (MGL from Trichomonas vaginalis) and a chemical model system of the reaction were utilized in order to investigate the cofactor-dependent activation of TFM and previously uninvestigated DFM (L-difluoromethionine). The differences in toxicity between TFM and DFM were evaluated against Escherichia coli expressing TvMGL1, as well as the intact human pathogen T. vaginalis. The relationship between the chemical structure of the reactive intermediates produced from the enzymatic processing of these analogues and their cellular toxicity are discussed.