Clostridioides difficile is the most common cause of healthcare-associated diarrhea. infection of the gastrointestinal tract with this Gram-positive, obligate anaerobe can lead to potentially lifethreatening conditions in the antibiotic-treated populace. new therapeutics are urgently needed to treat this infection and prevent its recurrence. Here, we screened two libraries from the National Cancer Institute, namely, the natural product set III library (117 compounds) and the approved oncology drugs set V library (114 compounds), against C. difficile. In the two libraries screened, 17 compounds from the natural product set III library and 7 compounds from the approved oncology drugs set V library were found to exhibit anticlostridial activity. The most potent FDA-approved drugs (mitomycin C and mithramycin A) and a promising natural product (aureomycin) were further screened against 20 clinical isolates of C. difficile. The anticancer drugs, mitomycin C (MIC 50 = 0.25 μg/ml) and mithramycin A (MIC 50 = 0.015 μg/ml), and the naturally derived tetracycline derivative, aureomycin (MIC 50 = 0.06 μg/ ml), exhibited potent activity against C. difficile strains. Mithramycin A and aureomycin were further found to inhibit toxin production by this pathogen. Given their efficacy, these compounds can provide a quick supplement to current treatment to address the unmet needs in treating C. difficile infection and preventing its recurrence.Clostridioides difficile is the leading cause of health care-associated diarrhea and mortality in the United States 1-4 . The Centers for Disease Control and Prevention (CDC) classified C. difficile as an urgent threat due to the immense suffering and death of thousands of patients each year. As per the 2017 estimates, the bacterium accounted for 223,900 cases with 12, 800 deaths and an attributable health care cost of $ 1 billion 5 . The characteristic manifestation of C. difficile-associated diarrhea (CDAD) ranges from mild diarrhea to pseudomembranous colitis, and toxic megacolon, and 1 out of every 11 patients aged 65 years or older dies within 30 days of diagnosis 6 .Antibiotic therapy remains a pivotal risk factor, as it aids the growth of C. difficile in the colon 7 . In its unperturbed state, the indigenous microflora of the colon serve as a host defense mechanism by providing resistance against colonization of the pathogen 8 . The use of antibiotics disrupts the host microflora, rendering individuals susceptible to C. difficile infection (CDI). In the absence of these gut microflora, following oral ingestion, the dormant C. difficile spores germinate, colonize the vacant nutrient niche in the gut, and release the enterotoxin TcdA and the cytotoxin TcdB 9,10 . These toxins A and B constitute the major virulence factors of the pathogen that damage the colonic epithelium triggering inflammatory responses that cause the range of symptoms associated with CDI 10,11 . Paradoxically, the standard treatment options for CDI includes oral administration of the antibiotics vancomycin or fidaxomi...