The Macrophage infectivity potentiator (Mip) protein is a virulence factor encoded by Gram-negative intracellular pathogens. Mip proteins have been shown to have a virulence-associated peptidyl-prolyl isomerase (PPIase) activity that is important for bacterial survival and pathogenesis. Therefore, inhibition of Mip potentially represents a novel target for antimicrobial therapies towards Gram-negative bacterial pathogens. A group of pipecolic acid-derived small molecules have been shown to inhibit recombinant Mip from Burkholderia pseudomallei (Bps). Using a protease coupled PPIase assay, we have demonstrated that these compounds also have broad-spectrum inhibitory properties against the recombinant Mips from Neisseria meningitidis and Coxiella burnetii. Importantly, these inhibitors reduce the cytotoxic effects and intracellular survival of these pathogens in mammalian cell lines. Co-crystal studies of inhibitor-BpsMip complexes have aided in developing compounds with improved affinity and potency that exhibit enhanced in vitro activity against the Gram-negative bacteria investigated in these studies.