Background: Exposure to antimicrobials is the major risk factor associated with Clostridium difficile infection (CDI). Paradoxically, treatment of CDI with antimicrobials remains the preferred option. To date, only three studies have investigated the antimicrobial susceptibility of C. difficile from Thailand, two of which were published in the 1990s. This study aimed to investigate the contemporary antibiotic susceptibility of C. difficile isolated from patients in Thailand. Methods: A collection of 105 C. difficile isolated from inpatients admitted at Siriraj Hospital in Bangkok in 2015 was tested for their susceptibility to nine antimicrobials via an agar incorporation method. Results: All isolates were susceptible to vancomycin, metronidazole, amoxicillin/clavulanate and meropenem. Resistance to clindamycin, erythromycin and moxifloxacin was observed in 73.3%, 35.2% and 21.0% of the isolates, respectively. The in vitro activity of fidaxomicin (MIC50/MIC90 0.06/0.25 mg/L) was superior to first-line therapies vancomycin (MIC50/MIC90 1/2 mg/L) and metronidazole (MIC50/MIC90 0.25/0.25 mg/L). Rifaximin exhibited potent activity against 85.7% of the isolates (MIC ≤0.03 mg/L), and its MIC50 (0.015 mg/L) was the lowest among all antimicrobials tested. The prevalence of multi-drug resistant C. difficile, defined by resistance to ≥3 antimicrobials, was 21.9% (23/105). Conclusions: A high level of resistance against multiple classes of antimicrobial was observed, emphasising the need for enhanced antimicrobial stewardship and educational programmes to effectively disseminate information regarding C. difficile awareness and appropriate use of antimicrobials to healthcare workers and the general public.