Eubacterial tRNA-guanine transglycosylase (TGT) is involved in the hypermodification of cognate tRNAs, leading to the exchange of G34 by preQ1 at the wobble position in the anticodon loop. Mutation of the tgt gene in Shigella flexneri results in a significant loss of pathogenicity of the bacterium due to inefficient translation of a virulence protein mRNA. Herein, we describe the discovery of a ligand with an unexpected binding mode. On the basis of this binding mode, three slightly deviating pharmacophore hypotheses have been derived. Virtual screening based on this composite pharmacophore model retrieved a set of potential TGT inhibitors belonging to several compound classes. All nine tested inhibitors being representatives of these classes showed activity in the micromolar range, two of them even in the submicromolar range.