Arctium minus (Hill) Bernh., commonly known as "Burdock", is a species within the Arctium genus of the Asteraceae family. Determining the optimum extraction conditions to obtain a concentrated extract with targeted active ingredients guides the most efficient use of natural products. Herein, ultrasound-assisted extraction (UAE) was optimized by using response surface methodology (RSM) to extract bioactive compounds from different organs of A. minus. Furthermore, phytochemical composition of extracts of the A. minus was investigated by using liquid chromatography-high resolution electrospray ionization mass spectrometry (LC-HRESIMS), antioxidant potential by using 2,2-diphenyl-1-picrylhydrazyl (DPPH), diazanium;3-ethyl-2-[(3-ethyl-6-sulfonato-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonate (ABTS), CUPRAC, and metal chelation assays, and cytotoxic activities by using human breast cancer cell line (MDA-MB-231) and hepatocellular carcinoma cancer cell line (HepG2), and compared against conventional methods; Soxhlet and maceration. The RSM was employed to investigate the influence of ultrasound power, extraction time, and extraction temperature on the antioxidant potential assessed by the DPPH free radical scavenging assay. In UAE of A. minus leaves, flowers, and branches, the conditions resulting in the minimum IC 50 values: 20 °C for 6 min at 50 W for leaves, 20 °C for 3 min at 100 W for flowers, and 20 °C for 3 min at 100 W for branches. Chlorogenic acid was identified as the major phenolic compound in the extracts obtained by UAE, with concentrations of 24,666.96 μg/g in leaves, 1054.92 μg/g in flowers, and 3,501.24 μg/g in branches. Flowers of A. minus had significantly higher levels of arctiin and arctigenin than those of leaves and branches. Extracts from leaves and flowers were more effective against MDA-MB-231 and HepG2 cancer cell lines than arctiin and arctigenin.