Hibiscus sabdariffa (Roselle) is in high demand worldwide due to its beneficial health properties owing to the polyphenols content, mainly in the flower calyx. The objective of this study was to find the best conditions (time and liquid: solid ratio) to extract polyphenols from Roselle using Ultrasound-Assisted Extraction (UAE) (40 kHz, 180 W), with ethanol how solvent; as well as determine the yield of phenols, anthocyanin, flavonoids, tannins, antioxidant activity (DPPH) and antigenotoxic effect (comet assay). A traditional solid-liquid extraction was applied as a reference. Extraction times of 40 and 60 min resulted in the highest polyphenols (13.019 mg GAE/g dry weight (dw)), flavonoids (4.981 CE/g dw), anthocyanins (1.855 mg Cya3GE/g dw), and tannins (0.745 CE/g dw) recoveries and an antioxidant activity (DPPH) of 74.58%. Extracts from white calyces contained similar amounts of phenols and flavonoids, but very little condensed tannins (0.049 CE/g dw) and practically no anthocyanins. Extracts from red and white calyces, showed antigenotoxic activity and repaired capacity of damage caused by mutagens in human lymphocytes.