Sea cucumbers are an important ingredient of traditional folk medicine in many Asian countries, which are well-known for their medicinal, nutraceutical, and food values due to producing an impressive range of distinctive natural bioactive compounds. Triterpene glycosides are the most abundant and prime secondary metabolites reported in this species. They possess numerous biological activities ranging from anti-tumour, wound healing, hypolipidemia, pain relieving, the improvement of nonalcoholic fatty livers, anti-hyperuricemia, the induction of bone marrow hematopoiesis, anti-hypertension, and cosmetics and anti-ageing properties. This study was designed to purify and elucidate the structure of saponin contents of the body wall of sea cucumber Holothuria
lessoni and to compare the distribution of saponins of the body wall with that of the viscera. The body wall was extracted with 70% ethanol, and purified by a liquid-liquid partition chromatography, followed by isobutanol extraction. A high-performance centrifugal partition chromatography (HPCPC) was conducted on the saponin-enriched mixture to obtain saponins with a high purity. The resultant purified saponins were analyzed using MALDI-MS/MS and ESI-MS/MS. The integrated and hyphenated MS and HPCPC analyses revealed the presence of 89 saponin congeners, including 35 new and 54 known saponins, in the body wall in which the majority of glycosides are of the holostane type. As a result, and in conjunction with existing literature, the structure of four novel acetylated saponins, namely lessoniosides H, I, J, and K were characterized. The identified triterpene glycosides showed potent antifungal activities against tested fungi, but had no antibacterial effects on the bacterium Staphylococcus aureus. The presence of a wide range of saponins with potential applications is promising for cosmeceutical, medicinal, and pharmaceutical products to improve human health.