Background <p><i>Colletotrichum gloeosporioides</i> infects the plant host via an appressorium, which generates turgor pressure by metabolizing certain internal compounds. The mechanical force created by the turgor pressure is required for the production of infection pegs that penetrate host epidermal cells. Thus, appressorium turgor pressure must be sufficient for <i>C. gloeosporioides</i> infection. The regulated metabolism of intracellular metabolites or compounds plays a key role in the process underlying increases in turgor pressure.</p> Results <p>In this study, we analyzed appressorial metabolites at different developmental stages using a non-targeted metabolomics approach. We identified 39 differentially accumulated metabolites associated with turgor pressure and classified them into six major groups: amino acids, fatty acids, phospholipids, glycerolipids, carbohydrates, and organic acids. Four notable metabolic pathways related to turgor pressure were identified: degradation of carbohydrates, degradation of lipids, amino acid (arginine) synthesis, and phospholipid metabolism. Moreover, the phospholipid metabolism may be important for the development of appressoria and the required increase in turgor pressure. Three representative inhibitors of phospholipid metabolism (Neomycin, Doxorubicin, and alexidine dihydrochloride) were selected to study the relationship of phospholipid metabolism on the development of appressorium and changes in turgor pressure in <i>C. gloeosporioides</i>. Neomycin was shown to be a potent inhibitor of <i>C. gloeosporioides</i> and could effectively control poplar anthracnose.</p> Conclusions <p>It was found that phospholipid metabolism is associated with the required increase in appressorium turgor. Our findings provide new insights into the mechanism underlying appressorium turgor pressure formation as well as potential targets for improving the control of <i>C. gloeosporioides.</i></p>