Clostridiodes (Clostridium) difficile is an anaerobic Gram-positive, spore-forming nosocomial, gastrointestinal pathogen causing C. difficile-associated disease with symptoms ranging from mild cases of antibiotic-associated diarrhea to fatal pseudomembranous colitis. We developed murine monoclonal antibodies (mAbs) specific for a conserved cell surface antigen, lipoteichoic acid (LTA), found on the surface of C. difficile. The mAbs were characterised in terms of their thermal stability, solubility, and their binding to LTA by surface plasmon resonance and competitive ELISA. Synthetic LTA molecules were prepared in order to better define the minimum epitope required to mimic the natural antigen and three repeat units of the polymer were required for optimal recognition. One of the murine mAbs was chimerised with human constant region domains and was found to recognise the target antigen identically to the mouse version. These mAbs may be useful as therapeutics (stand alone, in conjunction with known anti-toxin approaches, or as delivery vehicles for antibody drug conjugates targeting the bacterium), as diagnostic agents, and in infection control applications.