Abstract: This study aimed to investigate the antifungal activity of ginkgolic acids (GAs) against Rhizoctonia solani and preliminarily explore their underlying action mechanisms. The inhibitory effect of GAs on fungal growth was determined using the mycelial growth rate method. Scanning electron microscopy (SEM) was used to obverse morphological change of mycelia after GAs treatment. Additionally, the cell membrane permeability, malondialdehyde (MDA), and soluble protein content of GAs-treated mycelia were measured, along with the activities of catalase (CAT), superoxide dismutase (SOD), malate dehydrogenase (MDH), and succinate dehydrogenase (SDH). Transcriptome sequencing was employed to identify differentially expressed genes and conduct pathway enrichment analysis. Moreover, reverse transcription real-time quantitative PCR (RT-qPCR) was applied to verify the expression levels of key DEGs. The results showed that GAs exhibited significant antifungal activity against R. solani, with an EC₅₀ of 0.234 mg/mL. At a concentration of 0.300 mg/mL, the inhibition rate reached 60.59%, and the mycelia showed abnormal morphology, including shriveling, collapse, and disruption of branching structures. After 4 h of treatment with 0.300 mg/mL GAs, mycelium cell membrane permeability and MDA content significantly increased, while CAT, SOD, MDH, and SDH activities significantly decreased. Transcriptome analysis revealed that GAs induced 2018 DEGs, consisting of 906 upregulated and 1112 downregulated genes. These DEGs were significantly enriched in metabolic pathways, including fatty acid degradation, peroxisome, and steroid biosynthesis. RT-qPCR validation results are highly consistent with the transcriptome data. This study provides theoretical basis for the development and application of GAs as plant-derived antifungal agents.