SignificanceClostridioides difficileis the leading cause of hospital-acquired diarrhea worldwide. While TcdB, the primary virulence determinant ofC. difficile, is known to be required for the intestinal symptoms associated with infection, asymptomatic carriage of TcdB-positiveC. difficileis common, suggesting that specific host factors may influence disease severity. Here, we show that small molecules extracted from intestinal samples from mice and humans protect against low to moderate levels of TcdB; however, at higher concentrations of toxin, inhibition is overcome. We show that intestinal bile acids, which directly bind and inhibit TcdB, are responsible for the protection against TcdB. These findings demonstrate a role for intestinal bile acids in virulence and provide an anti-toxin approach to treat disease.