Background Anoectochilus roxburghii is a traditional Chinese medicine with potent medicinal activity owing to the presence of secondary metabolites, particularly flavonoids. A. roxburghii also maintains a symbiotic relationship with mycorrhizal fungi. Moreover, mycorrhizal fungi can induce metabolite synthesis in host plants. However, little is known about the role of mycorrhizal fungi in promoting the accumulation of flavonoid metabolites in A. roxburghii. Methods A. roxburghii and the isolated fungus Ceratobasidium sp. AR2 were cocultured. The portion of A. roxburghii above the medium treated with or without AR2 was studied by transcriptome and target metabolome analyses. Results AR2 promoted the growth and development of A. roxburghii. The contents of total flavonoid, rutin, isorhamnetin, and cyanidin-3-glucoside chloride were increased compared with those in uninoculated cultures. Transcriptome analysis suggested that 109 unigenes encoding key enzymes were potentially associated with changes in flavonoids. Quantitative real-time polymerase chain reaction of fourteen flavonoid-related unigenes showed that most flavonoid biosynthetic genes were significantly differentially expressed between inoculated and uninoculated plantlets. Conclusion The isolate AR2 could significantly promote the growth and development of A. roxburghii and the accumulation of flavonoids. Overall, our findings highlighted the molecular basis of the effects of mycorrhizal fungi on flavonoid biosynthesis in A. roxburghii and provided novel insights into methods to improve the yield and quality of A. roxburghii.