Phage-related ribosomal proteases (Prps) are essential for the assembly and maturation of the ribosome in Firmicutes, including the human pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Clostridium difficile. These bacterial proteases cleave off an N-terminal extension of a precursor of ribosomal protein L27, a processing step that is essential for the formation of functional ribosomes. This essential role of Prp in these pathogens has identified this protease as a potential antibiotic target. In this work, we determine the X-ray crystal structure of a covalent inhibition complex at 2.35 Å resolution, giving the first complete picture of the active site of a functional Prp. We also characterize the kinetic activity and screen for potential inhibitors of Prp. This work gives the most complete characterization of the structure and specificity of this novel class of proteases to date.