Current antibiotics for treating Clostridium difficile infections (CDI), that is, metronidazole, vancomycin and more recently fidaxomicin, are mostly effective but treatment failure and disease relapse remain as significant clinical problems. The shortcomings of these agents are attributed to their low selectivity for C. difficile over normal gut microflora and their ineffectiveness against C. difficile spores. This Letter reports that certain diarylacylhydrazones identified during a high-throughput screening/counter-screening campaign show selective activity against two Clostridium species (C. difficile and Clostridium perfringens) over common gut commensals. Representative examples are shown to possess activity similar to vancomycin against clinical C. difficile strains and to kill stationary-phase C. difficile cells, which are responsible for spore production. Structure–activity relationships with additional synthesised analogues suggested a protonophoric mechanism may play a role in the observed activity/selectivity and this was supported by the well-known protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) showing selective anti-Clostridium effects and activity similar to diarylacylhydrazones against stationary-phase C. difficile cells. Two diarylacylhydrazones were shown to be non-toxic towards human FaDu and Hep G2 cells indicating that further studies with the class are warranted towards new drugs for CDI.