BACKGROUND: Despite stringent infection control measures, Clostridioides difficile infection (CDI) remains a challenge in healthcare settings, partly due to overlooked transmission vectors such as toilet plume bioaerosols. AIM: To systematically quantify the risks associated with CDI transmission via toilet flushing and provide critical insights to inform CDI preventive strategies. METHODS: Impaction sampling was used to quantify airborne C. difficile post-flush and high-contact surfaces were swabbed to assess contamination levels, in a controlled toilet environment. A Quantitative Microbial Risk Assessment (QMRA) approach was then used to estimate the risk to subsequent users from contamination by a previously colonized individual. RESULTS: A single flush can release C. difficile into the air, with bioaerosol concentrations up to 29.50 ± 10.52 CFU/m CONCLUSION: The findings of this study necessitate a review of current toilet designs, public health policies and facility management practices to mitigate the overlooked risks of CDI transmission through toilet plume bioaerosols in healthcare settings. Additionally, this study lays a foundation for developing evidence-based interventions aimed at achieving substantial behavioural and infrastructural changes in infection control practices.