Frizzled 7 (FZD7) receptors have been shown to play a central role in intestinal stem cell regeneration and, more recently, in Clostridium difficile pathogenesis. Yet, targeting FZD7 receptors with small ligands has not been explored as an approach to block C. difficile pathogenesis. Here, we report the discovery of high affinity peptides that selectively bind to FZD7 receptors. We describe an integrated approach for lead optimization, utilizing structure-based rational design and directed evolution, to enhance the peptide binding affinity while still maintaining FZD7 receptor selectivity. This work yielded new peptide leads with picomolar binding constants to FZD7 as measured by biophysical methods. The new peptides block the interaction between C. difficile toxin B (TcdB) and FZD receptors and perturb C. difficile pathogenesis in epithelial cells. As such, our findings provide a proof of concept that targeting FZD receptors could be a viable pharmacological approach to protect epithelial cells from TcdB pathogenicity.