causes life-threatening diarrhea and is the leading cause of healthcare associated bacterial infections in the United States. During infection, releases the gut-damaging toxins, TcdA and TcdB, the primary determinants of disease pathogenesis and are therefore therapeutic targets. TcdA and TcdB contain a glycosyltransferase domain that uses UDP-glucose to glycosylate host Rho GTPases, causing cytoskeletal changes that result in a loss of intestinal integrity. Isofagomine inhibits TcdA and TcdB as a mimic of the oxocarbenium ion transition state of the glycosyltransferase reaction. However, sequence variants of TcdA and TcdB across the clades of infective continue to be identified and therefore, evaluation of isofagomine inhibition against multiple toxin variants are required. Here we show that Isofagomine inhibits the glycosyltransferase activity of multiple TcdB variants and also protects TcdB toxin-induced cell rounding of the most common full-length toxin variants. Further, isofagomine protects against induced mortality in two murine models of infection. Isofagomine treatment of mouse infection permitted recovery of the gastrointestinal microbiota, an important barrier to prevent recurring infection. The broad specificity of isofagomine supports its potential as a prophylactic to protect against induced morbidity and mortality.