The total synthesis and anticancer activity of several novel derivatives based on a dauer effect-inducing glycolipid are presented. A versatile and convergent synthesis was accomplished through stereospecific alpha-glycosylation, which produced di- and tri-rhamnoside daumone derivatives. Most of the synthetic derivatives possessed potent anticancer activity against human cancer cell lines. Daumone and deoxyrhamnose trisaccharides with amide side chains had the most potent anticancer activity among all other known glycolipids, with an effective concentration of 20 nM, which is comparable to that of doxorubicin. Conversely, acyclic and macrocyclic daumone derivatives had drastically decreased anticancer activity. Due to the high lipophilic nature of the novel glycolipid derivatives, we propose that the observed anticancer activity is due to their potential to inhibit cell differentiation and proliferation via interaction with the membranes of cancer cells.