Clostridioides difficile (C. difficile), which has historically denominated Bacillus difficile, was first isolated by Hall and O’Toole in fecal samples of newborns during the first 10 days of life in 1935. Isolation of C. difficile from animal fecal specimens is critical for understanding the susceptibility and virulence of circulating strains through in vitro and preclinical studies, helping clinicians and researchers understand the different outcomes of CDI in the host associated with the pathogen’s virulence. In this chapter, we will describe step-by-step procedures for isolating C. difficile from fecal specimens from animals with a focus on a method using taurocholate Cycloserine–Cefoxitin Fructose Agar (TCCFA) plates preceded by an alcohol shock.