Bioactivity guided isolation of oxypregnane-oligoglycosides (calotroposides) from the ethanolic extract of root bark of Calotropis gigantea (L.) Dryand. with purple flowers has been performed. Dereplication by NMR and LC-MS analysis confirmed the presence of oxypregnane-oligoglycosides (calotroposides) in the ethyl acetate fraction. One new (7) and six known calotroposides (1–6) were isolated from the most active ethyl acetate fraction of ethanolic extract of Calotropis gigantea and structure elucidation was accomplished by spectroscopic methods (IR, UV, MS and NMR). Isolated calotroposides were investigated for cytotoxic activity against six cancer cell lines (MDA-MB-231, MCF-7, LNCaP, HeLa, K562, and Caco-2) and a non-cancer cell line (HEK-293), where calotroposides 2, 4 and 5 showed highest cytotoxicity (IC50 8.49 ± 0.41 μM, 6.49 ± 0.15 μM and 9.65 ± 0.44 μM respectively) against MDA-MB-231. Conclusively, calotroposides 2, 4 and 5 have promising anticancer effect against aggressive breast cancer cells and can serve as leads for further structural exploration as anticancer moiety.