Multidrug resistance (MDR) is a major challenge in cancer treatment, and the breast cancer resistance protein (BCRP) is an important target in the search for new MDR-reversing drugs. With the aim of discovering new potential BCRP inhibitors, the crude extract of leaves of Eremophila galeata, a plant endemic to Australia, was investigated for inhibitory activity of parental (HT29_par) as well as BCRP-overexpressing HT29 colon cancer cells resistant to the chemothera-peutic SN-38 (HT29_SN38). The results showed that a fraction eluted with 40% acetonitrile on a solid-phase extraction column showed weak growth-inhibitory activity on HT29_SN38 cells when administered alone, but exhibited concentration-dependent growth inhibition when administered in combination with SN-38. The major constituent in this fraction was isolated and found to be 5,3′,5′-trihydroxy-3,6,7,4′-tetramethoxyflavone (2), which at a concentration of 25 μg/mL potentiated the growth-inhibitory activity of SN-38 to a degree comparable to that of the known BCRP inhibitor Ko143 at 1 μM. A dye accumulation experiment suggested that 2 inhibits BCRP, and docking studies showed that 2 binds to the same BCRP site as SN-38. These results suggest that 2 works synergistically with SN-38, with 2 acting as a BCRP efflux pump inhibitor and SN-38 as a topoisomerase-1 inhibitor.