Clostridioides difficile is, worldwide, the leading cause of hospital-acquired infection. Outbreaks are largely related to antibiotic exposure and contact contamination, but little is known about C. difficle infection (CDI) awareness in the nurse population. We conducted a cross-sectional survey to study Italian nurses, based on CDI guidelines. We recruited 200 nurses working in 14 Italian hospitals. Using a one-way analysis of variance of knowledge scores, female nurses (mean 9.67 (standard deviation ± 1.63), p = 0.03), and nurses with a higher level of university education (mean 9.79 (SD ± 1.67), p = 0.04) were demonstrated to have better knowledge about CDI. In addition, 92.5% (n = 184) of the sample declared that they did not have specific postgraduate training about CDI. Seventy-four percent (n = 149) of the respondents declared that they used procedures, protocols and guidelines about CDI in their workplace, but only 46.5% (n = 93) reported using C. difficile-specific bundles during their daily practice. In conclusion, our study highlights a lack of knowledge concerning CDI clinical guidelines among Italian nurses.