The effects of water and ethanolic (40 %, 70 %, and 96 %) extraction on the Rhodiola rosea L. phytochemical profile (HPLC analysis), stability during extract drying, potential bioaccessibility in simulated gastric and intestinal conditions, and cytotoxic activity against human colorectal adenocarcinoma cells (Caco-2 and HT-29 cell lines) were investigated. The phytochemical profile, extractability, and stability during extract processing depend on the solvent type. In general, compounds derived from dry extracts were characterized by higher bioaccessibility than those extracted from powdered plant material. In the case of salidroside, tyrosol, and rosavins, one of the highest bioaccessibilities (often about 100 %) were found for the 70 % ethanolic extract after gastric digestion. Furthermore, the 70 % ethanolic extract most effectively reduced the viability of Caco-2 cells (IC50 85.8 µg∙mL−1). The results suggest that golden root extracts, in particular 70 % ethanolic extract, seem to be promising supplements for the food industry.