Gastrodia elata Blume is a achlorophyllous orchid plant that has been used in traditional medicine to treat many human illnesses, such as vertigo, hemiplegia, and epilepsy. It contains three major pharmacological substances; gastrodin, ergothioneine, and vanillyl alcohol. Here we investigated the quantitative changes in those functional components by cultivation conditions and harvest times. The content of ergothioneine was different with cultivation areas, while gastrodin accumulated the higher levels than vanillyl alcohol regardless of cultivation areas. The smaller tubers contained the higher concentrations of gastrodin and the lower concentrations of ergothioneine than did the larger tubers. The cultivation under sun shade screen increased the concentrations of both gastrodin and vanillyl alcohol but decreased the levels of ergothioneine. We also determined the contents of functional substances depending on the harvest times. The concentration of gastrodin was higher in October than in April, while the level of ergothioneine was higher in April than in October. Interestingly, the use of sexually propagated seed tubers produced 1.9 times higher levels of gastrodin compared to vegetatively propagated seed tubers. In this study, we found that three major functional substances in G. elata tubers altered depending on various cultivation practices, which might be important to increase the economic values of G. elata tubers.