A series of coniugates bearing a 1,2,3,4-tetrahydroisoquinoline motif linked to substituted 7-hydroxy-2H-chromen-2-ones was synthesized and assayed through calcein-AM test in Madin-Darby Canine Kidney (MDCK) cells overexpressing P-glycoprotein (P-gp) and closely related multidrug resistance associated protein 1 (MRP1) to probe the interference with efflux mechanisms mediated by P-gp and MRP1, respectively. A number of substituents at C3 and C4 of coumarin nucleus along with differently sized and shaped spacers was enrolled to investigate the effects of focused structural modifications over affinity and selectivity. Linker length and flexibility played a key role in enhancing P-gp affinity as proved by the most potent P-gp modulator (3h, IC<sub>50</sub> = 70 nM). A phenyl ring within the spacer (3k, 3l, 3o) and bulkier groups (Br in 3r, Ph in 3u) at coumarin C3 led to derivatives showing nanomolar activity (160 nM < IC<sub>50</sub> < 280 nM) along with outstanding selectivity over MRP1 (SI > 350). Molecular docking calculations carried out on a human MDR1 homology model structure contributed to gain insights into the ligands' binding modes. Some compounds (3d, 3h, 3l, 3r, 3t, 3u) reversed MDR thereby restoring doxorubicin cytotoxicity when co-administered with the drug into MDCK-MDR1 cells.