Abstract Rose root biomass contains various bioactive compounds, including metabolites, which have been characterized for potential applications in the development of pharmaceuticals, natural cosmetics, and functional foods. This study aimed to determine how solvent selection influences the efficiency of ultrasound-assisted extraction (UAE) of rose root phytochemicals, as well as the biological activity and in vivo toxicity of the resulting extracts. Sixteen types of extracts were obtained using inexpensive, pharmaceutical-grade, and environmentally friendly solvents (methanol, acetone, and water). Their chemical profiles were characterized using liquid chromatography coupled with mass spectrometry (LC-APCI-MS/MS and LC-ESI-MS/MS). Samples’ antioxidant properties were assessed via the Oxygen Radical Absorbance Capacity (ORAC) assay, and the inhibition of lipase and α-glucosidase was evaluated through in vitro assays. The in vivo toxicity was tested using zebrafish embryo models. Employing a simple UAE protocol enabled the efficient elution of substantial quantities of highly bioactive triterpenes (e.g., tormentic, euscaphic, maslinic, betulinic, ursolic acids), polyphenols, and β-sitosterol. The methanol-water (3:1, v/v) extract showed the highest triterpene content, including euscaphic acid (19875 µg/g DW) and tormentic acid (5291.67 µg/g DW). Catechin was found to be the rose root dominating polyphenol (up to 30877.19 µg/g DW). The acetone-water (1:1, v/v) extract exhibited the most potent antioxidant and α-glucosidase inhibitory activities. All extracts demonstrated a favorable safety profile. These findings encourage further studies on the sustainable and large-scale recovery of rose root bioactive metabolites, indicating the perspectives for the effective valorization of this biomass into high-value ingredients or products.