Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhoea worldwide and is perpetuated by C. difficile spores; however, there are no cost-effective treatments that target spores or spore formation. The persistence of C. difficile spores results in recurrent CDI and costs $US1.5 billion yearly, indicating that anti-sporulation treatments are an urgent unmet healthcare need. Our published work has shown that the cephamycin antibiotic, cefotetan, can reduce C. difficile sporulation by inhibiting the spore-specific penicillin-binding protein (PBP), CdSpoVD, which is essential for spore synthesis. Of clinical relevance, co-treatment of cefotetan and the primary CDI treatment vancomycin, which does not target spores, prevented CDI relapse in mice. While cefotetan is a promising anti-sporulation candidate, our recent data has revealed that some C. difficile animal isolates have acquired an accessory spore-protein (CdSpoCR) that blocks the ability of cefotetan to reduce spore numbers, highlighting the need for anti-sporulation strategies that can target CdSpoCR strains. Here, we performed sporulation assays to determine if the inhibitor DL1 or the antibiotic cefmetazole could reduce sporulation relative to untreated cultures in a CdSpoCR+ animal isolate, and used protein profiling to identify their targets. DL1 reduced spore numbers completely by targeting CdSpoCR. Cefmetazole reduced spore numbers completely by targeting CdSpoVD and preventing the induction of CdSpoCR. Collectively, these results highlight the potential for DL1 or cefmetazole to overcome resistance to sporulation inhibition in a CdSpoCR+ strain. This inhibitor or its derivatives could improve CDI treatment by reducing spore numbers in a wider range of C. difficile isolates as well as other spore-forming pathogens for which no anti-sporulation treatments currently exist.