Legionella pneumophila, an opportunistic bacterial pathogen, is responsible for the mild-symptomatic Pontiac fever as well as the life-threatening Legionnaires' disease, which has a lethality rate of 10 %. The macrophage infectivity potentiator (Mip) was the first genetically identified virulence factor of L. pneumophila. This peptidyl-prolyl-cis/trans-isomerase is involved in the early stages of infection, tissue migration and enhancement of flagellation. Due to its important role for bacterial pathogenesis, inhibition of Mip could be a promising approach to improve the treatment of Legionellosis and reduce severe pneumonia. Six novel inhibitors, based on the immunosuppressive PPIase-binding protein FK506, were first tested in minimal inhibitory concentration and cytotoxicity assays and were later assessed in cell line infections with THP-1 and A549. While the macrolide FK506 evokes cytotoxic effects at concentrations of 50 μM, those novel inhibitors did not show cytotoxicity for lung epithelial cells in a resazurin-based assay. Five out of six compounds were functional inhibitors of bacterial growth and intracellular replication at a concentration of at least 12.5 μM. The best two candidates showed particularly high efficacy as they reduced the bacterial cell count during infection more than seven fold, similar to that of a L. pneumophila Δmip deletion strain. Interestingly, in previous studies derivatives of FK506 already showed additional off-target effects, which cannot be explained by Mip binding and may also play a role in this study. Thus, the novel non-immunosuppressive FK506 variants seem to be great candidates for a therapeutic application to reduce the bacterial proliferation during L. pneumophila infection, but also for future examination of not yet known off-target binding partners in L. pneumophila or the human host.