This study investigates the antifungal activity and mechanism of rosemary essential oil (REO) against *Colletotrichum gloeosporioides*, the primary pathogen causing walnut anthracnose. The research employs scanning electron microscopy (SEM), index determination, and transcriptome sequencing to evaluate the effects of REO on *C. gloeosporioides*. Key findings include: 1. **Antifungal Activity**: REO exhibited significant antifungal activity against *C. gloeosporioides*, with minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values of 15.625 μL/mL and 31.25 μL/mL, respectively. 2. **Morphological Changes**: SEM revealed that REO treatment caused severe deformation and shrinkage of *C. gloeosporioides* mycelium. 3. **Cell Wall and Plasma Membrane Effects**: REO increased chitinase activity, which decomposes fungal cell wall components, and decreased ergosterol content in the plasma membrane, leading to the release of intracellular contents. 4. **Energy Metabolism**: REO inhibited the tricarboxylic acid (TCA) cycle and energy metabolism by affecting key enzymes such as succinate dehydrogenase (SDH), malate dehydrogenase (MDH), ATPase, and phosphofructokinase (PFK). 5. **Transcriptome Sequencing**: Differential gene expression analysis showed that REO inhibited cell wall formation, genetic material synthesis, cell division, and differentiation in *C. gloeosporioides*. The results provide a comprehensive understanding of the efficacy and antifungal mechanism of REO against *C. gloeosporioides*, offering a theoretical basis for its potential use as a biological control agent in plant disease management.This study investigates the antifungal activity and mechanism of rosemary essential oil (REO) against *Colletotrichum gloeosporioides*, the primary pathogen causing walnut anthracnose. The research employs scanning electron microscopy (SEM), index determination, and transcriptome sequencing to evaluate the effects of REO on *C. gloeosporioides*. Key findings include: 1. **Antifungal Activity**: REO exhibited significant antifungal activity against *C. gloeosporioides*, with minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values of 15.625 μL/mL and 31.25 μL/mL, respectively. 2. **Morphological Changes**: SEM revealed that REO treatment caused severe deformation and shrinkage of *C. gloeosporioides* mycelium. 3. **Cell Wall and Plasma Membrane Effects**: REO increased chitinase activity, which decomposes fungal cell wall components, and decreased ergosterol content in the plasma membrane, leading to the release of intracellular contents. 4. **Energy Metabolism**: REO inhibited the tricarboxylic acid (TCA) cycle and energy metabolism by affecting key enzymes such as succinate dehydrogenase (SDH), malate dehydrogenase (MDH), ATPase, and phosphofructokinase (PFK). 5. **Transcriptome Sequencing**: Differential gene expression analysis showed that REO inhibited cell wall formation, genetic material synthesis, cell division, and differentiation in *C. gloeosporioides*. The results provide a comprehensive understanding of the efficacy and antifungal mechanism of REO against *C. gloeosporioides*, offering a theoretical basis for its potential use as a biological control agent in plant disease management.