Riboswitches are structured RNA domains that can bind directly to specific ligands and regulate gene expression. These RNA elements are located most commonly within the noncoding regions of bacterial mRNAs, although representatives of one riboswitch class have been discovered in organisms from all three domains of life. In several Gram-positive species of bacteria, riboswitches that selectively recognize guanine regulate the expression of genes involved in purine biosynthesis and transport. Because these genes are involved in fundamental metabolic pathways in certain bacterial pathogens, guanine-binding riboswitches may be targets for the development of novel antibacterial compounds. To explore this possibility, the atomic-resolution structure of a guanine riboswitch aptamer from Bacillus subtilis was used to guide the design of several riboswitch-compatible guanine analogues. The ability of these compounds to be bound by the riboswitch and repress bacterial growth was examined. Many of these rationally designed compounds are bound by a guanine riboswitch aptamer in vitro with affinities comparable to that of the natural ligand, and several also inhibit bacterial growth. We found that one of these antimicrobial guanine analogues (6-N-hydroxylaminopurine, or G7) represses expression of a reporter gene controlled by a guanine riboswitch in B. subtilis, suggesting it may inhibit bacterial growth by triggering guanine riboswitch action. These studies demonstrate the utility of a three-dimensional structure model of a natural aptamer to design ligand analogues that target riboswitches. This approach also could be implemented to design antibacterial compounds that specifically target other riboswitch classes.