n this study, an optimised high hydrostatic pressure process (600 MPa, 15 min, 55 and 65 °C) caused elimination of microbial levels in beetroot/carrot juice (80:20 v/v, pH 4.0) after 28 days of storage (4 °C) (psychrotrophic bacteria, yeast and mould counts below the limit of detection = 0 log cfu mL−1). Room temperature (20 °C) was insufficient for the complete elimination of psychrotrophic bacteria (0.9–2.0 log cfu mL−1 after 7–28 days of storage). No significant (P < 0.05) changes in the polyphenol content were observed (1.11 ± 0.13 in control vs. 1.13 ± 0.13 mg gallic acid mL−1 in samples treated at 65 °C). No significant (P < 0.05) changes in the antioxidant, antiradical activity and biological properties of the high-pressure processing (HPP)-treated juice were observed in the studies involving CCD 841 CoTr and HT29 cell lines. The HPLC-MS analysis confirmed increase in isobetalain, decarboxybetalain and betalain content (by 100%, 14% and 10%) and loss of vulgaxanthin I (by 65%) after HPP at 65 °C. The innovative character of this paper stems from the comprehensive quality assessment (especially experiments involving cell lines) of high-quality, safe beetroot–carrot juice that retains a major part of its bioactive compounds.