Spores are produced by many organisms as the result of a survival mechanism, triggered under several environmental stresses. These are multi-layered structures, one of which is a peptidoglycan layer known as the cortex. This structure contains muramic-δ-lactams that are synthesized by at least two enzymes, CwlD and PdaA. This study focuses on the spore cortex of Clostridium difficile, a Gram-positive spore-forming, toxin-producing anaerobic pathogen that can colonize the intestinal tract of humans, considered as the leading cause of antibiotic-associated diarrhea. The cortex of the C. difficile 630∆erm strain was analyzed using UHPLC coupled to HRMS. This analysis revealed that C. difficile cortex differs from B. subtilis cortex. Amongst these differences, the muramic-δ-lactams represented 24% in C. difficile, compared to 50% in B. subtilis. CD630_14300 and CD630_27190 were identified as the genes encoding the C. difficile N-deacetylases PdaA1 and PdaA2, responsible for muramic-δ-lactam synthesis. In the pdaA1 mutant only 0.4% of all muropeptides carry a muramic-δ-lactam modification, and no muramic-δ-lactam were found in the cortex of the double mutant pdaA1-pdaA2. Surprisingly, our results suggest a much broader impact for muramic-δ-lactams in C. difficile compared to previously characterized model organisms, such