Rosemary essential oil (REO) effectively inhibits the growth of Colletotrichum gloeosporioides, the pathogen causing walnut anthracnose. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of REO were determined to be 15.625 μL/mL and 31.25 μL/mL, respectively. Scanning electron microscopy (SEM) showed that REO treatment caused severe deformation of fungal mycelium. The activity of chitinase, which breaks down fungal cell wall components, increased, while the content of ergosterol, a key component of the fungal plasma membrane, decreased. This led to the release of intracellular components such as nucleic acids, soluble proteins, and reducing sugars, altering extracellular electrical conductivity. Additionally, the activities of enzymes involved in energy metabolism, including succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and ATPase, decreased, while phosphofructokinase (PFK) activity increased. Transcriptome analysis revealed that REO disrupts the cell wall and membrane, inhibits genetic material synthesis, and affects cell division and differentiation. These findings highlight the antifungal mechanism of REO against C. gloeosporioides and support its potential as a natural biocontrol agent. The study provides a theoretical basis for the development of REO as an effective fungicide.Rosemary essential oil (REO) effectively inhibits the growth of Colletotrichum gloeosporioides, the pathogen causing walnut anthracnose. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of REO were determined to be 15.625 μL/mL and 31.25 μL/mL, respectively. Scanning electron microscopy (SEM) showed that REO treatment caused severe deformation of fungal mycelium. The activity of chitinase, which breaks down fungal cell wall components, increased, while the content of ergosterol, a key component of the fungal plasma membrane, decreased. This led to the release of intracellular components such as nucleic acids, soluble proteins, and reducing sugars, altering extracellular electrical conductivity. Additionally, the activities of enzymes involved in energy metabolism, including succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and ATPase, decreased, while phosphofructokinase (PFK) activity increased. Transcriptome analysis revealed that REO disrupts the cell wall and membrane, inhibits genetic material synthesis, and affects cell division and differentiation. These findings highlight the antifungal mechanism of REO against C. gloeosporioides and support its potential as a natural biocontrol agent. The study provides a theoretical basis for the development of REO as an effective fungicide.