Shigella spp. are among the main causative agents of acute diarrheal illness and claim more than 1 million lives per year worldwide. There are multiple bacterial genes that control the pathogenesis of Shigella, but the virF gene may be the most important. This gene, located on the primary pathogenicity island of Shigella, encodes VirF, an AraC-family transcriptional activator that is responsible for initiating the pathogenesis cycle in Shigella. We have previously shown that it is possible to attenuate the virulence of Shigella flexneri via small molecule inhibition of VirF. In this study, we probed the mechanism of action of our small molecule inhibitors of VirF. To enable these studies, we have developed a homologous and efficient expression and purification system for VirF and have optimized two different in vitro VirF-DNA binding assays. We have determined that one of our HTS hit compounds inhibits VirF binding to DNA with a calculated Ki similar to the effective doses seen in our transcriptional activation and virulence screens. This is consistent with inhibition of DNA binding as the mechanism of action of this hit compound. We have also screened 15 commercially sourced analogs of this compound and deduced an initial SAR from the approximately 100-fold range in activities. Our four other HTS hit compounds do not inhibit DNA binding and yet they do block VirF activity. This suggests that multiple agents with different molecular mechanisms of inhibition of VirF could be developed. Pursuing hits with different mechanisms of action could be a powerful approach to enhance activity and to circumvent resistance that could develop to any one of these agents.