Sixteen cardenolide triglycosides (1–16) were isolated from stems, twigs, and leaves of Nerium oleander. Among them, 3β-O-(4-O-gentiobiosyl-d-diginosyl)-7β,8-epoxy-14-hydroxy-5β,14β-card-20(22)-enolide, named cardenolide B-3 (16), was isolated from natural sources for the first time. The in vitro anti-inflammatory activities of compounds 1–16 were examined on the basis of inhibitory activity against the induction of intercellular adhesion molecule-1 (ICAM-1). Compounds 1–5 were active at an IC50 value of less than 7 μM. The cytotoxic activity of isolated compounds was evaluated against three human cell lines: normal human fibroblast cells (W-38), malignant tumor cells induced from WI-38 (VA-13), and human liver tumor cells (HepG2). Compounds 1–5 were active toward WI-38 cells; compounds 1, 3, and 5 were active toward VA-13 cells; and compounds 1–5 were active toward HepG2 cells at IC50 values of less than 10 μM. The multidrug-resistant (MDR) cancer-reversal activity of compounds 1–16 was evaluated on the basis of the amount of calcein accumulated in MDR human ovarian cancer 2780AD cells in the presence of each compound. Compounds 13 and 14 showed significant effects on calcein accumulation.