Clostridioides difficile infection (CDI) is the fifth leading cause of death from nonmalignant gastrointestinal disease in the United States. The contribution of resistance to C. difficile-active antibiotics to the outcomes of CDI is unclear. We evaluated the antimicrobial susceptibility of C. difficile isolates in a U.S. hospital and determined associations of clinical variables and binary toxin positivity with antibiotic resistance. C. difficile spores were cultured from fecal specimens of adult patients with CDI for genotyping and antimicrobial susceptibility assay (for clindamycin CLI, fidaxomicin FDX, metronidazole MTZ, moxifloxacin MXF, tigecycline TGC, and vancomycin VAN). Electronic medical records were reviewed for clinical data extraction. Ninety-seven of 130 (75%) fecal samples grew toxigenic C. difficile in culture. Most of the isolates were (80.4%), and 18.6% and 1% were and , respectively. Susceptibility to VAN, MTZ, FDX, TGC, MXF, and CLI was 96%, 94%, 100%, 100%, 8%, and 79%, respectively. Six isolates, all positive and belonging to the 027 ribotype, were resistant to VAN and/or MTZ. Higher MICs were found in isolates with a mutation in the VAN-related resistance gene , but not . In addition, isolates exhibited higher MICs of VAN, MTZ, TGC, CLI, and MXF compared to strains. Patients with greater intestinal inflammation or severe disease were more likely to be infected with strains. Decreased susceptibility to antibiotics is not directly associated with either severe or recurrent CDI. However, antimicrobial susceptibility of C. difficile is decreased in strains positive for the binary toxin gene.