Breast cancer resistance protein (BCRP), a member of the ATP-binding cassette transporter family, plays a key role in the efflux of various drugs and is linked to multidrug resistance in cancer therapy. Flavonoids, particularly those with a flavonol backbone, have shown promise as inhibitors of BCRP activity; however, their specific inhibitory effects are not fully understood. This study explored the inhibitory effects of 77 flavonols on BCRP and identified 22 compounds with significant inhibitory activity. Among them, 14 flavonols had half-maximal inhibitory concentrations (IC) values below 5 µM, effectively reversing BCRP-mediated resistance to SN-38 in vitro. Molecular docking analysis revealed key interactions between flavonols and BCRP, including π-stacking, hydrogen bonding, and hydrophobic interactions. Structural modifications, including hydroxylation and methylation, enhanced the binding affinity of the flavonols. In vivo studies with 3,4'-dimethoxyflavone and 3,6,3',4'-tetramethoxyflavone resulted in increased systemic exposure to sulfasalazine, a known BCRP substrate. These findings provide mechanistic insights into flavonol-BCRP interactions, suggesting their potential to enhance drug exposure and efficacy. Future research should focus on clinical applications to explore the therapeutic potential of these flavonols for improved drug responses.