AbstractChemotherapy is a widely used strategy to treat cancer, a disease that causes millions of deaths each year. However, its efficacy is reduced by the overexpression of ABC transporters, which are proteins that expel the drugs used in chemotherapy and involved in the multidrug resistance (MDR). Glycolipids have been identified as potential inhibitors of ABC transporters. Algae of the genus Sargassum contain high levels of glycolipids, making them a promising therapeutic alternative against the MDR phenotype. Sargassum filipendula glycolipids were obtained by exhaustive maceration with chloroform/methanol, purified by column and thin layer chromatography, and then characterized by FTIR, NMR, and LC‐MS. Cell viability by PI labeling and inhibition of ABC transporters were analyzed by flow cytometry. Assessment of resistance reversal was determined by MTT assay. Ten sulfoquinovosylglycerol‐type compounds were found, and six of them are reported for the first time. In particular, moiety 4 (GL‐4) showed strong and moderate inhibitory activity against ABCC1 and ABCB1 transporters respectively. Treatment of GL‐4 in combination with the antineoplastic drug vincristine sensitized Lucena‐1 cell model to drug and reversed the MDR phenotype. This is the first report of glycolipids isolated from S. filipendula capable of inhibiting ABC transporters and thus overcoming acquired drug resistance.