Strains of Trichoderma harzianum are well-known producers of bioactive secondary metabolites and have a beneficial effect on plants. However, to the best of our knowledge, the effect of the commonly used pesticides on the activity of this fungus is not yet investigated. Therefore, in the present study, the effect of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on the lipidome and selected extracellular compounds synthesized by T. harzianum IM 0961 was examined. It was observed that the herbicide 2,4-D caused changes in the lipid composition of the mycelium and that the herbicide exhibited lipophilic properties. In addition, the herbicide disturbed the phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio and increased membrane permeability. The higher amount of cardiolipin CL 72:7 and the lower amount of CL 72:8 could have been associated with a decreased ratio of 18:2 and 18:1 fatty acids in the herbicide-treated samples. Moreover, in the presence of 2,4-D, an increased lipid peroxidation (twofold), as well as a higher content of oxylipin (9-HODE and 13-HODE) and phosphatidic acid (PA), was noted, confirming that 2,4-D induced lipid peroxidation in the mycelium. The herbicide also exerted its toxic effect on the production of 14-aminoacid peptaibols and two compounds, harzianic acid and t22-azaphilone, exhibiting antibiotic and plant growth-promoting activity. During proteomic analysis, the synthesis of some proteins, such as calcineurin-like phosphoesterase metallophosphatases (MPPs), which modulate the properties of cell walls, was found to be inhibited by the herbicide. These presented findings may be of significant value in understanding the effect of 2,4-D on the activity of T. harzianum. Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.