Intestinal bile acids are known to modulate the germination and growth of Clostridioides difficile. Here we describe a role for intestinal bile acids in directly binding and neutralizing TcdB toxin, the primary determinant of C. difficile disease. We show that individual primary and secondary bile acids reversibly bind and inhibit TcdB to varying degrees through a mechanism that requires the combined oligopeptide repeats region to which no function has previously been ascribed. We find that bile acids induce TcdB into a compact “balled up” conformation that is no longer able to bind cell surface receptors. Lastly, through a high-throughput screen designed to identify bile acid mimetics we uncovered nonsteroidal small molecule scaffolds that bind and inhibit TcdB through a bile acid-like mechanism. In addition to suggesting a role for bile acids in C. difficile pathogenesis, these findings provide a framework for development of a mechanistic class of C. difficile antitoxins.