Alachlor is a 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 has 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 employs 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 culture growth and glucose consumption rate. At the same time, the rates of glycolysis and the tricarboxylic acid (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 xenobiotic presence 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 resulted in a deeper insight into microorganism strategy toward xenobiotic biodegradation.