The outer membrane (OM) of Gram-negative bacteria acts as a physical barrier protecting Gram-negative species from harmful environmental influences. At the same time it facilitates the import of nutrients, the export of proteins, the passage of signaling molecules and also harbors proteins associated with virulence. Transmembrane traffic particularly is facilitated by membrane-integral proteins. In order to insert these into the OM, an essential oligomeric membrane-associated protein complex, the beta-barrel assembly machinery (BAM) is required. Being essential for the biogenesis of outer membrane proteins (OMPs) the BAM and also periplasmic chaperones (termed the OMP biogenesis machinery as an entity herein) may serve as attractive targets to develop novel antimicrobial or antiinfective agents. We aimed to elucidate which proteins belonging to the OMP biogenesis machinery have the most important function in granting bacterial fitness, facilitating biogenesis of dedicated virulence factors and determination of overall virulence. To this end we used the enteropathogen Yersinia enterocolitica (Ye) as a model system. We individually knocked out all non-essential components of the BAM (BamB, C and E) as well as the periplasmic chaperones DegP, SurA and Skp. In summary, we found that the most profound phenotypes were produced by the loss of BamB or SurA with both knockouts resulting in significant attenuation or even avirulence of Ye in a mouse infection model. Thus, both BamB and SurA are promising targets for the development of new antimicrobials in the future.