Withanolides are steroidal lactones commonly found in plants of the Solanaceae family that have significant medicinal value. In this study, three withanolides extracted from Iochroma arborescens leaves were isolated and characterized. These included withaphysalin F (3: ) and two newly identified epimeric compounds: 18R- and 18S-O-methyl-withaphysalin F (1: and 2: ). Their structures were elucidated by NMR, IR, MS, CD, and X-ray diffraction analysis, and their potential against cell proliferation and migration was investigated. The cytotoxic assay revealed activity against different tumor and non-tumor cell lines. (18S)-O-methyl-withaphysalin F (2: ) presented cell death effects after at least 6 hours of exposure. MDA-MB-231 cells were exposed to 0.06 and 0.6 µM of (18S)-O-methyl-withaphysalin F (2: ), and reductions in cell adhesion, migration, and clonogenicity were observed. Morphological analysis revealed negative regulation in filopodia, salience, and roughness, as well as alterations in cellular microarchitecture. These results provide clues as to the effects of (18S)-O-methyl-withaphysalin F (2: ), allowing new molecular modifications to improve potency and selectivity and increase our antineoplastic arsenal.