Alachlor is an herbicide that is widely used worldwide to protect plant crops against broadleaf weeds and annual grasses. However, due to its endocrine-disrupting activity, its application had been banned in the European Union. As described in our earlier work, Paecilomyces marquandii is a microscopic fungus capable of alachlor removal by N-acetyl oxidation. Our current work uses proteomics and metabolomics to gain a better understanding of alachlor biodegradation by the microscopic fungus P. marquandii. The data revealed that the addition of alachlor reduced the culture growth and glucose consumption rates. Moreover, the rates of glycolysis and the tricarboxylic acids (TCA) cycle increased during the initial stage of growth, and there was a shift toward the formation of supplementary materials (UDP-glucose/galactose) and reactive oxygen species (ROS) scavengers (ascorbate). Proteomic analysis revealed that the presence of xenobiotics resulted in a strong upregulation of enzymes related to energy, sugar metabolism and ROS production. However, the unique overexpression of cyanide hydratase in alachlor-containing cultures may implicate this enzyme as the key protein involved in the alachlor biodegradation pathway. The characterization of P. marquandii-mediated alachlor removal in terms of cell structure and function provides a deeper insight into the strategies of microorganisms toward xenobiotic biodegradation.