The anaerobic bacterium Clostridioides difficile represents one of the most problematic pathogens, especially in hospitals. Dysbiosis has been proven to largely reduce colonization resistance against this intestinal pathogen. The beneficial effect of the microbiota is closely associated with the metabolic activity of intestinal microbes such as the ability to transform primary bile acids into secondary ones. However, the basis and the molecular action of bile acids (BAs) on the pathogen are not well understood. We stressed the pathogen with the four most abundant human bile acids: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and lithocholic acid (LCA). Thin layer chromatography (TLC), confocal laser scanning microscopy (CLSM), and electron microscopy (EM) were employed to track the enrichment and destination of bile acids in the bacterial cell. TLC not only revealed a strong accumulation of LCA in C. difficile, but also indicated changes in the composition of membrane lipids in BA-treated cells. Furthermore, morphological changes induced by BAs were determined, most pronounced in the virtually complete loss of flagella in LCA-stressed cells and a flagella reduction after DCA and CDCA challenge. Quantification of both, protein and RNA of the main flagella component FliC proved the decrease in flagella to originate from a change in gene expression on transcriptional level. Notably, the loss of flagella provoked by LCA did not reduce adhesion ability of C. difficile to Caco-2 cells. Most remarkably, extracellular toxin A levels in the presence of BAs showed a similar pattern as flagella expression. That is, CA did not affect toxin expression, whereas lower secretion of toxin A was determined in cells stressed with LCA, DCA or CDCA. In summary, the various BAs were shown to differentially modify virulence determinants, such as flagella expression, host cell adhesion and toxin synthesis. Our results indicate differences of BAs in cellular localization and impact on membrane composition, which could be a reason of their diverse effects. This study is a starting point in the elucidation of the molecular mechanisms underlying the differences in BA action, which in turn can be vital regarding the outcome of a C. difficile infection.