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Clostridioides difficile is an anaerobic, spore-forming pathogen that causes serious toxin-mediated enteric disease in humans. In addition to antimicrobial resistance, biofilm and spore formation play key roles in the persistence of C. difficile in the gut, as well as in the transmission and relapse of the disease. In this study, the antimicrobial potential of dill seed essential oil on the planktonic growth of C. difficile clinical strains (isolated from stool specimens of hospitalized patients with diarrhea and confirmed Clostridioides difficile infection (CDI)) was investigated, along with its effect on biofilm and spore formation. The results showed varying degrees of antimicrobial activity, ranging from strong to weak, depending on the strain, with concentrations ranging from 0.08 to 40 mg/ml. The essential oil (EO) at concentrations of 2xMIC and MIC significantly reduced biofilm production in 89% and 84% of the tested strains, respectively. Spore formation was also significantly reduced when treated with 0.5xMIC and MIC of EO. Considering the anticlostridial activity of the dill seed EO, along with its inhibition of biofilm production and sporulation, this natural product is an excellent candidate for supplementary treatment of CDI. [ABSTRACT FROM AUTHOR]