Roots and leaves of Carlina acaulis L. are still used in ethnomedicine in many European countries; however, the limited occurrence of the plants and protection of this species necessitate a search for alternative ways for obtaining this plant material. In this study, in vitro cultures, hydroponic cultures, and field cultivation were applied to obtain the C. acaulis plant material. Its quality was evaluated using antioxidant activity tests and high performance liquid chromatography analysis. Our study showed that the antioxidant activity and the content of chlorogenic and 3,5-di-caffeoylquinic acid in roots of plants cultivated in hydroponics and field conditions were comparable. However, the amount of carlina oxide was significantly higher in plants from the field. The flavonoid content in leaves obtained from both cultivation systems was at the same level; however, the antioxidant activity and the content of the investigated metabolites were higher in the soil cultivation system. The callus line exhibited high differentiation in phytochemical compositions depending on the treatments and medium compositions.