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Clostridioides difficile is the leading cause of hospital-associated diarrhea and has remained a consistent threat for older patients and those with comorbidities or vulnerabilities. The high rates of treatment failure and recurrence, along with the decreased effectiveness of first-line treatments highlight the urgent need for the development of new anti-C. difficile agents. α-mangostin is a natural compound isolated from the edible mangosteen fruit pericarps that has known antimicrobial activity. α-mangostin is poorly absorbed from the gastrointestinal tract (GIT), which is ideal for treatment of CDI to accumulate at the site of infection at concentrations capable of clearing C. difficile. We found that α-mangostin was as potent as the standard-of-care vancomycin, inhibiting a diverse panel of C. difficile strains at a concentration range of 0.5–2 µg/mL. It exhibited rapid bactericidal activity, completely clearing C. difficile in vitro within 2 hours, surpassing vancomycin and fidaxomicin. Additionally, α-mangostin’s anti-C. difficile activity was not affected by the high C. difficile inoculum. To further understand its mechanism, we investigated α-mangostin’s membrane disruption activity by assessing the leakage of DNA and ATP post-exposure. α-mangostin resulted in a significant leakage of DNA and ATP indicating that its anti-C. difficile activity is mediated by the bacterial cell membrane disruption. Collectively, these findings demonstrate that α-mangostin possesses desirable characteristics for a promising anti-C. difficile which merits further investigation.