The type III secretion (TTS) system is used by several animal and plant pathogens to deliver effector proteins into the cytosol of the eukaryotic target cell as a strategy to evade the defense reactions elicited by the infected organism. The fact that these systems are highly homologous implies that novel antibacterial agents that chemically attenuate the pathogens via a specific interaction with the type III secretion mechanism can be identified. A number of small organic molecules having this potential have recently been identified (A. M. Kauppi, R. Nordfelth, H. Uvell, H. Wolf-Watz, and M. Elofsson, Chem. Biol. 10:241-249, 2003). Using different reporter gene constructs, we showed that compounds that belong to a class of acylated hydrazones of different salicylaldehydes target the TTS system of Yersinia pseudotuberculosis. One of these compounds, compound 1, was studied in detail and was found to specifically block Yop effector secretion under in vitro conditions by targeting the TTS system. In this respect the drug mimics the well-known effect of calcium on Yop secretion. In addition, compound 1 inhibits Yop effector translocation after infection of HeLa cells without affecting the eukaryotic cells or the bacteria. A HeLa cell model that mimics in vivo conditions showed that compound 1 chemically attenuates the pathogen to the advantage of the eukaryotic cell. Thus, our results show proof of concept, i.e., that small compounds targeting the TTS system can be identified, and they point to the possible use of TTS inhibitors as a novel class of antibacterial agents.