Anoectochilus roxburghii (Wall.) Lindl. (A. roxburghii) is an increasingly popular medicinal herb. Arbuscular mycorrhiza (AM) fungi, known for their symbiotic relationships with plant roots, enhance nutrient uptake and disease resistance in host plants. However, their specific regulatory mechanisms in A. roxburghii are not fully understood. In this study, Fujian A. roxburghii was inoculated with the AM fungus Glomus intraradices, and successful root colonization was observed. Following AM fungal colonization, there was a significant upregulation of photosynthesis-related genes in the stems, accompanied by improved canopy phenotypes and root architecture. Consequently, AM-inoculated plants exhibited increased fresh and dry biomass, as well as elevated levels of polysaccharides and flavonoids. Additionally, the incidence of Fusarium oxysporum-induced stalk rot was reduced in AM-inoculated plants. Analysis of defense-related enzymes indicated that AM-inoculated plants exhibited a rapid and robust response to pathogen infection, mitigating oxidative stress. Transcriptomic analysis revealed significant upregulation of genes associated "Fatty acid degradation", "MAPK signaling pathway-plant", and "Plant-pathogen interaction", suggesting their involvement in enhanced disease resistance. A regulatory network centered on ACX1 and calmodulin, involving multiple transcription factors such as WRKY, bHLH, ERF, NAC, and HSF, was implicated in defense responses. These findings demonstrated the beneficial effects of AM fungi on yield, quality, and disease resistance in A. roxburghii, providing a theoretical foundation for its cultivation and genetic improvement.