Despite an alarming increase in morbidity and mortality caused by multidrug-resistant bacteria, the number of antibiotics available to efficiently combat them is dwindling. Consequently, there is a pressing need for new drugs, preferably with novel modes of action to avert the problem of cross-resistance. Several new targets have been proposed, including proteins essential in the protein secretion pathway such as the type I signal peptidase (SPase), indispensable for the release of the signal peptide during secretion of Sec- and Tat-dependent proteins. The type I SPase is considered to be an attractive target because it is essential, substantially different from the eukaryotic counterpart, and its active site is located at the outer leaflet of the cytoplasmic membrane, permitting relatively easy access to potential inhibitors. A few SPase inhibitors have already been identified, but their suitability as drugs is yet to be confirmed. An overview is given on the currently known SPase inhibitors, how they can give valuable information on the structural, biochemical and target validation aspects of the SPases, the approaches to identify them, and their future potential as drugs.