Autophagy is an evolutionarily conserved process to protect cells against unfavorable conditions. Increasing evidence confirmed that autophagy provides metabolic products for tumor cells, and thereby reduced the sensitivity of tumor cells to chemotherapeutic drugs. In this regard, autophagy inhibition may represent a potential approach for cancer therapy. We screened a series of pulchinenosides or analogues and found that some of them (e.g. pulchinenoside D, E2 and E4) strongly inhibited autophagic flux in human breast cancer cells, which is evidenced by increased levels of LC3II turnover and p62 proteins. The structure-activity relationship analysis revealed that 2-4 sugar unites of a single oligosaccharide chain linked to C-3 are required for the activity, suggesting that the property of surfactant plays a pivotal role for pulchinenosides in the inhibition of autophagic flux. The mechanistic study demonstrated that the active pulchinenosides disrupted lipid rafts of lysosomal membrane, increased lysosomal pH, suppressed lysosomal cathepsins activation and lead to lysosomal dysfunction. Furthermore, the active pulchinenosides decreased the production of intracellular ATP and thereby caused cellular energy depletion, induced p62-medicated ubiquitinated protein aggregation and eventually promoted apoptosis in breast cancer cells. The active pulchinenosides synergistically enhanced the anticancer activity of chemotherapeutic agents, e.g. camptothecin and 5-fluorouracil, in in vitro and in vivo tumor models. These results reveal a prominent autophagy-inhibitory activity of pulchinenosides and implicate their potential application in breast cancer therapy.