Adherence of uropathogenic Escherichia coli to host tissue is mediated by pili, which are hair-like protein structures extending from the outer cell membrane of the bacterium. The chaperones FimC and PapD are key components in pilus assembly since they catalyse folding of subunits that are incorporated in type 1 and P pili, respectively, and also transport the subunits across the periplasmic space. Recently, compounds that inhibit pilus biogenesis and interfere with chaperone–subunit interactions have been discovered and termed pilicides. In this paper NMR spectroscopy was used to study the interaction of different pilicides with PapD and FimC in order to gain structural knowledge that would explain the effect that some pilicides have on pilus assembly. First relaxation-edited NMR experiments revealed that the pilicides bound to the PapD chaperone with mM affinity. Then the pilicide–chaperone interaction surface was investigated through chemical shift mapping using ¹⁵N-labelled FimC. Principal component analysis performed on the chemical shift perturbation data revealed the presence of three binding sites on the surface of FimC, which interacted with three different classes of pilicides. Analysis of structure–activity relationships suggested that pilicides reduce pilus assembly in E. coli either by binding in the cleft of the chaperone, or by influencing the orientation of the flexible F1–G1 loop, both of which are part of the surface by which the chaperone forms complexes with pilus subunits. It is suggested that binding to either of these sites interferes with folding of the pilus subunits, which occurs during formation of the chaperone–subunit complexes. In addition, pilicides that influence the F1–G1 loop also appear to reduce pilus formation by their ability to dissociate chaperone–subunit complexes.