AimsThere is a need for novel treatments for Clostridium difficile infection (CDI). Antibacterial flavonoids are part of a large family of polyphenol phytochemicals with a long history of use in ethnomedicine, but are unexamined against Cl.difficile. We explored their anti-difficile properties. Methods and ResultsAnti-difficile activities were determined for several naturally occurring flavonoids, olympicin A and synthetic 4-chromanone and chalcone analogues. With the exception of olympicin A, most naturally occurring phytochemicals tested were poorly active. Diversified synthetic flavonoids resembling olympicin A retained anti-difficile activity, suggesting olympicin A could act as a pharmacophore to obtain novel agents. They also demonstrated concentration-dependent killing of logarithmic and stationary phase cultures and reduced sporulation and toxin production. Olympicin A and some synthetic flavonoids dissipated the bacterial transmembrane potential. Interestingly, mutants could only be selected with the analogue 207 at a frequency of 10(-9). ConclusionsBased on the potent anti-difficile properties of olympicin A and modified flavonoids, further exploration of this class of phytochemicals is warranted. Significance and Impact of the StudyClostridium difficile infection is a major problem in developed countries. These studies point to there being an avenue for optimizing plant-derived flavonoids, and related antibacterial phytochemicals, as nature-inspired approaches to treat CDI.