Multidrug-resistant Gram-negative (GN) infections for which there are few available treatment options are increasingly common. The development of new antibiotics for these pathogens is challenging because of the inability of most small molecules to accumulate inside GN bacteria. Using recently developed predictive guidelines for compound accumulation in Escherichia coli, we have converted the antibiotic Ribocil C, which targets the flavin mononucleotide (FMN) riboswitch, from a compound lacking whole-cell activity against wild-type GN pathogens into a compound that accumulates to a high level in E. coli, is effective against Gram-negative clinical isolates, and has efficacy in mouse models of GN infections. This compound allows for the first assessment of the translational potential of FMN riboswitch binders against wild-type Gram-negative bacteria.