<p>P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are typical ABC efflux transporters that play important physiological and pharmacological roles. At the blood–brain barrier (BBB), P-gp and BCRP function cooperatively as the main efflux transporters, hindering the entry of drugs into the brain. Therefore, dual inhibition of P-gp and BCRP is needed to deliver drugs effectively to the brain. This study sought potential dual P-gp/BCRP inhibitors to determine their ability to enhance brain penetration of the anticancer drug mitoxantrone (MX) and thereby improve its therapeutic efficacy against brain cancer. Candidate compounds for dual P-gp/BCRP inhibitors were extracted using in silico algorithms. The dual P-gp/BCRP inhibitory activity of 75 extracted candidates was investigated through MX accumulation studies in breast cancer cell lines overexpressing P-gp (MCF-7/ADR) and BCRP (MCF-7/MX100). The 5 compounds selected as final candidates were CDK 4/6 inhibitor IV, BX795, foretinib, BI-D1870, and CGP60474. Each of these 5 candidates increased MX accumulation and reversed MX resistance in MCF-7/ADR and MCF-7/MX100 cells. Additionally, they increased MX permeability across the BBB in an in vitro model. In situ brain perfusion studies showed that CDK 4/6 inhibitor IV, BX795, and CGP60474 improved the brain delivery of MX in rats. Moreover, in a mouse brain tumor model, CDK 4/6 inhibitor IV and BX795 potentiated the anticancer effect of MX against brain cancer, leading to a considerable reduction in tumor burden. In conclusion, potential dual P-gp/BCRP inhibitors were discovered through in silico screening and verified through in vitro and in vivo studies. CDK 4/6 inhibitor IV was the most effective dual P-gp/BCRP inhibitor candidate for enhancing the brain penetration of an anticancer drug for the treatment of brain tumors.</p>