Ametryn is a representative of a class of s-triazine herbicides absorbed by plant roots and leaves and characterized as a photosynthesis inhibitor. It is still in use in some countries in the farming of pineapples, soybean, corn, cotton, sugar cane or bananas; however, due to the adverse effects of s-triazine herbicides on living organisms use of these pesticides in the European Union has been banned. In the current study, we characterized the biodegradation of ametryn (100 mg L-1) by entomopathogenic fungal cosmopolite Metarhizium brunneum. Ametryn significantly inhibited the growth and glucose uptake in fungal cultures. The concentration of the xenobiotic drops to 87.75 mg L-1 at the end of culturing and the biodegradation process leads to formation of four metabolites: 2-hydroxy atrazine, ethyl hydroxylated ametryn, S-demethylated ametryn and deethylametryn. Inhibited growth is reflected in the metabolomics data, where significant differences in concentrations of L-proline, gamma-aminobutyric acid, L-glutamine, 4-hydroxyproline, L-glutamic acid, ornithine and L-arginine were observed in the presence of the xenobiotic when compared to control cultures. The metabolomics data demonstrated that the presence of ametryn in the fungal culture induced oxidative stress and serious disruptions of the carbon and nitrogen metabolism. Our results provide deeper insights into the microorganism strategy for xenobiotic biodegradation which may result in future enhancements to ametryn removal by the tested strain.