<p>Multidrug resistance efflux pumps are among the primary contributors to chemotherapy failure. Therefore, the identification of novel inhibitors with greater specificity and efficacy remains a critical objective. Here the effects of harmane, harmine, and norharman were investigated on the activity of P-gp and MRP1 transporters in the EPG85.257RDB and A2780 cell lines. The lipophilicity of these compounds and their potential interactions with the transporters were assessed via molecular docking. Cytotoxicity, changes in the IC₅₀ of daunorubicin, and efflux pump activity were evaluated using MTT assays and flow cytometry. Additionally, the effects of these alkaloids on gene expression were measured by real-time PCR and western blotting. Molecular docking revealed that all three alkaloids interact with the drug binding sites of P-gp and MRP1, suggesting potential roles as substrates or inhibitors. Among the tested compounds, harmane, having the highest lipophilicity, also showed the strongest cytotoxicity. Co-treatment with the alkaloids reduced the IC₅₀ of daunorubicin in both cell lines, indicating enhanced chemosensitivity. Flow cytometry results further confirmed that harmane and harmine significantly impaired the efflux functions of both P-gp and MRP1 transporters. However, these compounds had limited and inconsistent effects on gene and protein expression of the pumps. In conclusion, these alkaloids enhanced the sensitivity of EPG85.257RDB and A2780 cells to daunorubicin, likely through inhibition of drug efflux activity. These findings highlight β-carboline alkaloids as promising candidates for combination therapy in overcoming multidrug resistance in cancer.</p>