Spore-forming bacteria encompass a diverse range of genera and species, including important human pathogens. Due to their inherent resistance and unique structure, spores are highly problematic in health care settings, in the food industry and as potential bioterrorism agents, resulting in high economic costs and disease burden. Clostridium difficile is a spore-forming pathogen and global health threat, responsible for outbreaks of hospital-acquired diarrhoea. Even though spores are critical in C. difficile disease transmission and recurrence, current treatments are ineffective against spores. We identified a group of compounds (DL01, DL02, DL03) which have inhibitory effects on C. difficile sporulation in vitro and in vivo. Excitingly, we found that co-treatment of mice with DL02 and the current standard-of-care, vancomycin, prevented disease recurrence. TEM imaging suggests that these compounds block early stages of sporulation. Through mass spectrometry, mutagenesis and binding studies, we determined that the compounds target spore-specific proteins. This family of compounds directly impacting on sporulation could contribute to current regimes in treating C. difficile infections. Importantly, this anti-sporulation study could significantly advance drug development for other important spore-forming pathogens.