Coumarin and its derivatives have emerged as promising candidates in drug discovery. While the activity of coumarins as anticancer agents with different biological targets has been thoroughly investigated, reports on the potential of coumarins in the inhibition of thioredoxin reductase (TrxR) are still scarce. We focus on the design and synthesis of 3,4-unsubstituted coumarin analogues with systematic incorporation of substituents at the fifth to eighth positions of coumarin, which allowed definitive structure-activity relationship analysis to be conducted. In the obtained library, the substitution at the sixth position of the coumarin core with an aromatic or a cyclopropyl group turned out to be more activity enhancing. A bulky aromatic substituent with a large CF3 group encourages ligand alignment in a manner that enables covalent bond formation with the catalytic TrxR1 residue, according to the docking results. Our observations indicate that the activity of a series of coumarin analogues towards thioredoxin reductase 1 (TrxR1) is dependent on the nature (size and electronic effect) and the position of the substituent and more importantly - the accessibility of the Michael acceptor functionality. Several compounds (with at least 90% inhibition of the rat TrxR1 enzyme at 200 μM concentration) were further examined in in vitro cell-based assays to assess the cytotoxic effects on various cancer cell lines. The analogue 6-(4-(trifluoromethyl)phenyl)-2H-chromen-2-one was selected as the lead compound for further optimization. The results presented herein pave the way for the development of the next generation of coumarin-based TrxR1 inhibitors, where modification of the Michael acceptor moiety and incorporation of different aryl substituents at the sixth position of the coumarin core are planned.