AbstractCandida gut colonization and yeast biofilm production capacity were investigated, by means of XTT reduction assay, in Clostridioides difficile infected (CDI) patients, in non-CDI diarrheic patients, and in healthy donors in two different time periods (2013–2015 and 2018–2019 respectively). Candida gut colonization was significantly (p < 0.001) associated to C. difficile infection, and to patients infected with hypervirulent C. difficile strains bearing the tcdC deletion at nucleotide 117 (p = 0.0003). Although there was not a prevalent yeast species in CDI patients, C. albicans was the species significantly (p < 0.001) associated to both the infections sustained by the non-hypervirulent C. difficile strains and those caused by the hypervirulent strain (p = 0.001). The biofilm production by the yeasts isolated from the CDI patients and from non-CDI diarrheic patients did not differ significantly. However, a significantly (p = 0.007) higher biofilm production was observed in the Candida strains, particularly C. albicans, isolated from healthy donors compared to that of the yeasts cultured from CDI patients. Seasonal occurrence was observed in the isolation rate of CDI and non-CDI diarrheic cases (p = 0.0019), peaking in winter for CDI patients and in spring for non-CDI diarrheic patients. Furthermore, seasonality emerged in the gut colonization by Candida of CDI patients in the winter. It seems, therefore, that the reduced capacity of biofilm production by Candida strains isolated from CDI patients might have a role in the development of C. difficile infection, probably facilitating the spread of the bacteria into the gut thus amplifying their pathogenic action.