Clostridium difficile, a leading cause of hospital-acquired infection, possesses a dense surface layer (S-layer) that mediates host-pathogen interactions. The key structural components of the S-layer result from proteolytic cleavage of a precursor protein, SlpA, into high- and low-molecular-weight components. Here we report the discovery and optimization of the first inhibitors of this process in live bacteria and their application for probing S-layer processing. We also describe the design and in vivo application of activity-based probes that identify the protein Cwp84 as the cysteine protease that mediates SlpA cleavage. This work provides novel chemical tools for the analysis of S-layer biogenesis and for the potential identification of novel drug targets within clostridia and related bacterial pathogens.