Beetroot (Beta vulgaris L.) is an important root vegetable crop and a valuable food source of micronutrients and bioactive constituents. In this study, the bioaccessibility of minerals, organic acids, and betacyanins in beetroot powder during simulated gastrointestinal digestion was investigated, as well as the antioxidant activity of the final fractions of each phase of the process. Mineral elements were analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES), organic acids by ultra-fast liquid chromatography with photodiode array detection (UFLC-PDA), and betacyanins by liquid chromatography with diode-array detection and mass spectrometry (HPLC-DAD-ESI/MSn). The oxidative hemolysis inhibition assay was used to assess the ex vivo antioxidant activity. The bioaccessibility of minerals at the end of gastrointestinal digestion ranged from 43 to 65%, depending on the mineral element. Among these, Mg was the most bioaccessible, while Ca and Fe had the lowest bioaccessibility. For organic acids, a major release during digestion was observed for quinic acid. It was also found that betanin (the major betalain in beetroot) was highly unstable during the digestion process, probably due to its hydrophilic nature, which agreed with the significant (p < 0.05) decrease in antioxidant/antihemolytic activity. These results suggest that beetroot antioxidant compounds are unstable under gastrointestinal conditions, and could be useful for future development of novel and more stable beetroot food formulations.