We have designed structure-based ligands for the guanidine-II riboswitch that bind with enhanced affinity, exploiting the twin binding sites created by loop–loop interaction. We synthesized diguanidine species, comprising two guanidino groups covalently connected by C_n linkers where n = 4 or 5. Calorimetric and fluorescent analysis shows that these ligands bind with a 10-fold higher affinity to the riboswitch compared to guanidine. We determined X-ray crystal structures of the riboswitch bound to the new ligands, showing that the guanidino groups are bound to both nucleobases and backbone within the binding pockets, analogously to guanidine binding. The connecting chain passes through side openings in the binding pocket and traverses the minor groove of the RNA. The combination of the riboswitch loop–loop interaction and our novel ligands has potential applications in chemical biology.