Clostridioides difficile infection (CDI) is mediated by the cytotoxins TcdA and/or TcdB. The reliable detection of these toxins using enzyme immunoassays (EIA) could be affected by toxin stability. We assessed the toxin stability in stool samples spiked with TcdA or TcdB and in stool samples from CDI patients under transport conditions. In the spiked samples, the concentration of TcdB decreased faster than TcdA within 72 h. In contrast, toxin levels in stool samples from CDI patients remained stable within 24 h, while viable C. difficile slightly increased. In conclusion, toxin instability did not affect C. difficile toxin detection in our study.