Metalaxyl (MY) is a water soluble synthetic acylanilide fungicide widely used in agriculture practices to control phytopathogenic fungi, but it elicits environmental pollution to underground water as well as human health. The safe and effective removal of agrochemical pollutants from contaminated field sites is performed by some soil and aquatic microorganisms. However, limited work has been done focusing on the intermediate metabolites' residues produced and enzymes involved in the biodegradation process of fungicides. The degradation ability of the isolated aquatic fungus Aspergillus wentii of the MY as a sole carbon source in only sterilized distilled water was evaluated in vitro after 15 days, reaching high levels of biodegradation at 68%. Furthermore, the possible catabolic degradation pathways of MY based on the detected metabolites beside the enzymes involved in the biodegradation process were established. Moreover, a TripleTOF 5600 + LC-MS/MS was employed to identify the enzymes involved in the biodegradation process of the fungicide. The results revealed that CYP450 is responsible for metabolism of MY. It could be concluded that Aspergillus wentii enzymes have a prospective potential to eliminate a wide spectrum of toxic environmental pollutants, such as MY. To our knowledge, this study is the first report that Aspergillus wentii CYP 450 could catalyze the degradation of metalaxyl.