Candida albicans is one of the most common causes of fungal infections in humans with a significant mortality rate. However, the factors involved in C. albicans gastrointestinal (GI) colonization remain unclear. We hypothesize that secondary bile acids have direct antifungal activity against C. albicans and may play a critical role in maintaining GI colonization resistance against C. albicans. In this study, we investigated the effect of secondary bile acids including lithocholic acid (LCA) and deoxycholic acid (DCA) on C. albicans growth and morphogenesis. Results indicate that LCA and DCA at in vivo cecal micelle concentrations inhibit C. albicans growth in vitro. Interestingly, LCA and DCA also significantly inhibited the germ tube, hyphae and biofilm formation in C. albicans. In addition, pre-treatment of C. albicans with LCA and DCA significantly reduced the percentage of C. albicans cells attached to a colon cancer cell line. Collectively, our results demonstrate that secondary bile acids play an important role in controlling the growth and morphological switching of C. albicans. Results from this study demonstrate that secondary bile acid possess direct antifungal activity against C. albicans, explaining a potential mechanism for gastrointestinal colonization resistance against C. albicans.