lostridium difficile (C. difficile) is a problematic Gram positive bacterial pathogen causing moderate to severe gastrointestinal infections. Based on a lead binaphthyl-tripeptide dicationic antimicrobial, novel mono-, di- and tri-peptidomimetic analogues targeting C. difficile were designed and synthesized incorporating one, two or three D-configured cationic amino acid residues, with a common 1,2,3-triazole ester isostere at the C-terminus. Copper- and ruthenium-click chemistry facilitated the generation of a 46 compound library for in vitro bioactivity assays, with structure–activity trends over the largest compound subset revealing a clear advantage to triazole-substitution with a linear or branched hydrophobic group. The most active compounds were dicationic-dipeptides where the triazole was substituted with a 4- or 5-cyclohexylmethyl or 4,5-diphenyl moiety, providing MICs of 4 μg mL−1 against three human isolates of C. difficile. Further biological screening revealed significant antimicrobial activity for several compounds against other common bacterial pathogens, both Gram positive and negative, including S. aureus (MICs ≥2 μg mL−1), S. pneumoniae (MICs ≥1 μg mL−1), E. coli (MICs ≥4 μg mL−1), A. baumannii (MICs ≥4 μg mL−1) and vancomycin-resistant E. faecalis (MICs ≥4 μg mL−1).