Macrophage infectivity potentiator (MIP) proteins, found in pro- and eukaryotic pathogens, influence microbial virulence, host cell infection, pathogen replication, and dissemination. MIPs share an FKBP (FK506 binding protein)-like prolyl--isomerase domain, making them attractive targets for inhibitor development. We determined high-resolution crystal structures of and MIPs in complex with fluorinated pipecolic acid inhibitors. The inhibitor binding profiles in solution were compared across , , and MIPs using H, N, and F NMR spectroscopy. Demonstrating the versatility of fluorinated ligands for characterizing inhibitor complexes, F NMR spectroscopy identified differences in ligand binding dynamics across MIPs. EPR spectroscopy and SAXS further revealed inhibitor-induced global structural changes in homodimeric MIP. This study demonstrates the importance of integrating diverse methods to probe protein dynamics and provides a foundation for optimizing MIP-targeted inhibitors in this structurally conserved yet dynamically variable protein family.