PriA helicase catalyzes the initial steps of replisome reloading onto repaired DNA replication forks in bacterial DNA replication restart pathways. We have used a high-throughput screen to identify a small molecule inhibitor of PriA-catalyzed duplex DNA unwinding. The compound, CGS 15943, targets Neisseria gonorrhoeae PriA helicase with an IC(50) of 114 ± 24 μM. The PriA helicase of Escherichia coli is also inhibited, although to a lesser extent than N. gonorrhoeae PriA. CGS 15943 decreases rates of PriA-catalyzed ATP hydrolysis and reduces the affinity with which PriA binds DNA. Steady-state kinetic data indicate that CGS 15943 inhibits PriA through a mixed mode of inhibition with respect to ATP and with respect to DNA, indicating that it binds to a site on PriA that participates in both substrate binding and catalysis. Inhibitor binding constants derived from steady-state kinetic experiments reveal that CGS 15943 has the highest binding affinity for the PriA·PriB·ATP complex, intermediate binding affinity for the PriA·PriB·DNA complex, and the lowest binding affinity for the PriA·PriB·DNA·ATP complex, suggesting that PriA assumes different conformations in each of these complexes. We propose that CGS 15943 binds to PriA at a site distinct from the DNA and primary ATP binding sites, perhaps at PriA's weak nucleotide binding site, and induces a conformational change in PriA that renders it less catalytically proficient or prevents conformational changes in PriA that are necessary for ATP hydrolysis and duplex DNA unwinding.