Alachlor is an herbicide that is widely used worldwide to protect plant crops against broadleaf weedsand annual grasses. However, due to its endocrine-disrupting activity, its application had been bannedin the European Union. As described in our earlier work, Paecilomyces marquandii is a microscopic funguscapable of alachlor removal by N-acetyl oxidation. Our current work uses proteomics and metabolomicsto 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 consump-tion rates. Moreover, the rates of glycolysis and the tricarboxylic acids (TCA) cycle increased duringthe 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 analy-sis revealed that the presence of xenobiotics resulted in a strong upregulation of enzymes related toenergy, sugar metabolism and ROS production. However, the unique overexpression of cyanide hydratasein alachlor-containing cultures may implicate this enzyme as the key protein involved in the alachlorbiodegradation pathway. The characterization of P. marquandii-mediated alachlor removal in terms of cellstructure and function provides a deeper insight into the strategies of microorganisms toward xenobioticbiodegradation.This study was supported by the grant of the National Science Centre, Poland (Project No. UMO-2011/01/B/NZ9/02898)