ABSTRACT As bacterial pathogens develop resistance against most currently used antibiotics, novel alternatives for treatment of microbial infectious diseases are urgently needed. Targeting bacterial virulence functions in order to disarm pathogens represents a promising alternative to classical antibiotic therapy. Type IV secretion systems, which are multiprotein complexes in the cell envelope that translocate effectors into host cells, are critical bacterial virulence factors in many pathogens and excellent targets for such “antivirulence” drugs. The VirB8 protein from the mammalian pathogen Brucella was chosen as a specific target, since it is an essential type IV secretion system component, it participates in multiple protein-protein interactions, and it is essential for the assembly of this translocation machinery. The bacterial two-hybrid system was adapted to assay VirB8 interactions, and a high-throughput screen identified specific small-molecule inhibitors. VirB8 interaction inhibitors also reduced the levels of VirB8 and of other VirB proteins, and many of them inhibited virB gene transcription in Brucella abortus 2308, suggesting that targeting of the secretion system has complex regulatory effects in vivo . One compound strongly inhibited the intracellular proliferation of B. abortus 2308 in a J774 macrophage infection model. The results presented here show that in vivo screens with the bacterial two-hybrid assay are suited to the identification of inhibitors of Brucella type IV secretion system function.