Spirochetes are especially invasive bacteria that are responsible for several human diseases, including Lyme disease, periodontal disease, syphilis, and leptospirosis. Spirochetes rely on an unusual form of motility based on periplasmic flagella (PFs) to infect hosts and evade the immune system. The flexible hook of these PFs contains a post-translational modification in the form of a lysinoalanine (Lal) cross-link between adjacent subunits of FlgE, which primarily comprise the hook. Lal cross-linking has since been found in key species across the phylum and involves residues that are highly conserved. The requirement of the Lal cross-link for motility of the pathogens Treponema denticola (Td) and Borreliella burgdorferi (Bb) establish Lal as a potential therapeutic target for the development of antimicrobials. Herein, we present the design, development, and application of a NanoLuc-based high-throughput screen that was used to successfully identify two structurally related Lal cross-link inhibitors (hexachlorophene and triclosan) from a library of clinically approved small molecules. A structure–activity relationship study further expanded the inhibitor set to a third compound (dichlorophene), and each inhibitor was demonstrated to biochemically block autocatalytic cross-linking of FlgE from several pathogenic spirochetes with varied mechanisms and degrees of specificity. The most potent inhibitor, hexachlorophene, alters Lal cross-linking in cultured cells of Td and reduces bacterial motility in swimming plate assays. Overall, these results provide a proof-of-concept for the discovery and development of Lal-cross-link inhibitors to combat spirochete-derived illnesses.