The synthesis and inhibitory activity against DNA gyrase of a series of diphenic acid monohydroxamides 4a-f are described. A protocol of two biological assays showed conclusively that inhibition occurs specifically at the DNA-DNA gyrase complex and is not attributable to nonspecific inhibition. In the enzyme assays, 4c was potent as the prototypical quinolone, nalidixic acid (1), with an IC50 value of 58.3 micrograms/mL compared to 52 micrograms/mL for 1. MIC activity against bacterial strains showed a systematic drop for all compounds relative to 1. For compounds 4c-e, the addition of PMBN produced dramatic increases in MIC activity indicating that activity is likely to be related to membrane transport. Molecular modeling of 4a indicates that the diphenic acid monohydroxamides can bind to the DNA-DNA gyrase complex in a similar fashion as that hypothesized for the quinolone series according to the hypothesis suggested by Shen et al. but may not self-associate by pi-pi stacking. In contrast to the quinolone series, as the diphenic acid monohydroxamides are shown by molecular mechanics minimizations to be nonplanar, they may present novel approaches for chemotherapeutic intervention with a potential for decreased side effects.