Anoectochilus formosanus, commonly known as "Jewel Orchid", is a Chinese folk medicine used to treat hypertension, diabetes, and heart disease. The existence of A. formosanus is currently threatened by habitat loss, human and animal consumption, etc. The highly potent medicinal activity of A. formosanus is due to its secondary metabolites, especially kinsenosides and flavonoids. This orchid also has a unique mycorrhizal relationship. Most adult orchids rely on endophytes for mineral nutrition and have complex interactions with them, which are related to plant growth, yield and changes in secondary metabolites. This study investigated the promoting role of F-23 fungus (genus Mycena) on the biomass and contents of kinsenosides and flavonoids of A. formosanus in pot culture. The following were observed after 10 weeks of symbiotic cultivation: increased shoot height, shoot dry weight, and leaf numbers by 16.6%, 31.3%, and 22.5%, respectively; increased contents of kinsenosides, isorhamnetin-3-O-β-D-rutinoside, and isorhamnetin-3-O-β-D-glucopyranoside by 85.5%, 226.1%, and 196.0%, respectively; some hyphae in epidermal cells dyed red and/or reddish brown by safranine; and, significantly reduced number of starch grains in cortical cells. Moreover, F-23 fungus significantly improved the kinsenoside and flavonoid contents of A. formosanus. These findings supported the reports that endophytes can alter the production of secondary metabolites in their plant hosts, although further physiological, genetic and ecological analyses are warranted.