Two-partner secretion (TPS) systems are amongst the most widely conserved secretion systems across Gram-negative pathogens and consist of a TpsB family outer membrane-embedded protein which secretes a cognate exoprotein. The exoproteins can perform a myriad of virulence functions (e.g.: adhesion, toxins, immune modulation, etc.). Despite their incredible importance in bacterial infectious disease, TpsB inhibitors are yet to be discovered. Here we aimed to design the first TPS inhibitors by focusing on the protein ‘FhaC’, a well-characterized archetype of the TpsB family which secretes the exoprotein ‘FhaB’ that is required for the colonization of the lower respiratory tract by Bordetella spp leading to Whooping Cough and diseases in livestock. We designed a peptide called “P1-CPP” that we hypothesized would bind FhaC. We tested P1-CPP in an assay in which an E. coli strain producing FhaBC was treated. Strikingly, we observed that the peptide potently inhibited FhaB secretion (IC50=0.69uM) and caused the presecretory form of the exoprotein to accumulate inside the bacteria. Importantly, P1-CPP treatment also prevented FhaB secretion from Bordetella bronchiseptica and reduced adherence of the bacteria to CHO epithelial cells in vitro. Isothermal calorimetry experiments showed stable binding of P1-CPP to purified FhaC substrate-binding domain with a predicted affinity (Kd) of ~1.7uM which was further supported by molecular dynamics simulations. Our results strongly support that our inhibitor functions as a competitive inhibitor of FhaC as its mechanism of action and provides a proof of concept for TpsB family inhibitors.