Six previously undescribed withanolides, of which three had a normal glycosylated skeleton (Jaborosalactones 49-51) and three had a sativolide skeleton (12-O-methyl jaborosalactone 52, jaborosalactone 53, and 12-O-methyl jaborosalactone 53), were isolated from the aerial parts of Jaborosa riojana. In addition, eleven known compounds, featuring normal, sativolide, and trechonolide skeletons, were isolated. The structures of all compounds were fully characterized using a combination of spectroscopic techniques including multinuclear and multidimensional NMR and mass spectrometry. The in vitro antiproliferative activities of the major isolated compounds were evaluated against A549, HBL-100, HeLa, SW1573, T-47D, and WiDr human solid tumor cancer cell lines. Some withanolides showed antiproliferative activity comparable to cisplatin. The monitoring of cell cultures with continuous live-cell imaging indicated that the compounds induced apoptosis after 10 h of treatment.