ABCB1 is an active transporter physiologically expressed in pharmacologically important tissue barriers and frequently associated with the development of multidrug resistance in cancer cells. Dietary polyphenols involve a chemically related, but extremely diverse group of compounds abundant in plant derived food. Previous research demonstrated that several polyphenols are substrates or inhibitors of certain ABC transporters. Our experiments revealed that certain dietary polyphenols found in sour cherry and other red berries interact with human ABCB1as substrates or inhibitors. When applied alone, these polyphenols did not induce complete ABCB1 inhibition. However, combined treatments with low concentration of verapamil and various polyphenols such as QUR, naringenin or ellagic acid brought about a practically complete inhibition of ABCB1. Similarly, the combined application of QUR and C3S also induced a synergistic ABCB1 inhibitory effect, which could be exploited in chemotherapy protocols targeting multidrug-resistant tumors. To understand the molecular details of the interaction of C3S and QUR we carried out in silico ligand docking studies and MD simulations. These experiments revealed that C3S and QUR may bind simultaneously to the complex substrate-binding pocket of ABCB1. The most favourable binding poses were obtained when the bulkier C3S molecule bound to the central substrate binding site and the smaller QUR molecule occupied the “access tunnel”. Binding free energy calculations demonstrated that the simultaneous binding of two C3S or two QUR molecules to the complex substrate-binding pocket of ABCB1 is energetically less favourable compared to the C3S–QUR combination. The additive or synergistic transport inhibitory effects observed with polyphenol–verapamil combinations highlight a potential risk of drug–drug interactions on the level of ABCB1. These interactions necessitate more caution when consuming dietary polyphenols alongside chemotherapy regimens involving ABCB1 substrate or inhibitor drugs. Collectively, the integration of wet lab data and in silico studies enhances our understanding of the molecular mechanisms underlying ligand–ABCB1 interactions and pave the way for more efficient inhibition strategies of ABCB1 to overcome multidrug resistance.