Metsulfuron methyl (MM) is a sulfonylurea herbicide used worldwide for the control of weeds in cereal crops. In a previous study, three Trichoderma strains (T5, T6, and T7) capable of using MM as a sole carbon and energy source were isolated. In this study, the three strains were identified as Trichoderma harzianum using genetic markers, and the transformation of MM by the T. harzianum strains was quantified using spectrophotometry. Solutions of different phytotoxic doses of MM were incubated with plugs of mycelia of the Trichoderma strains and the resulting mixtures were used to assess MM detoxification. The toxicity of the degradation products was tested with a bioassay using pre-germinated seeds of Lens culinaris Medik. and mycelia. Strain T7 was more efficient in transforming MM at higher concentrations than the T5 and T6 strains. In the bioassay, T5 showed the best performance at higher MM doses. We conclude that both T5 and T7 strains are promising for further studies regarding treatment or amelioration of MM contaminated soils.