<p>Plants and their multiple secondary metabolites represent one of the main sources of potentially new pharmaceuticals. Digoxin, digitoxin, and ouabain are a group of cardenolides produced by plants of the genera <i>Digitalis</i>, <i>Strophanthus</i>, and <i>Acokenthera</i> that are used in the treatment and management of congestive cardiac insufficiency, arrhythmias, and heart failure. Alternatively, several cardenolides such as oleandrin, isolated from <i>Nerium oleander</i> L., Apocynaceae, have been reported to show antitumor and antiviral activity <i>in vitro</i>, particularly against COVID-19. As part of our search for new cardenolides with antiviral activity from plant species of the Apocynaceae family in the Yucatecan flora, our examination of the leaf extract of <i>N. oleander</i> resulted in the isolation and identification of 8-hydroxi-digitoxigenin-3-<i>O</i>-β-<span>D</span>-diginoside, a new bioactive cardenolide showing a lower cytotoxic activity than that of oleandrin when tested using the Madin-Darby canine kidney cell line. Molecular docking analysis showed that the new cardenolide has a lower molecular affinity to the Na⁺/K⁺ ATPase protein than oleandrin, suggesting that this difference in affinity between the two cardenolides could explain their differences in cytotoxic activity, despite their having similar chemical structures and polarities.</p> Graphical Abstract <p></p>