In the current study, we show that biofilm formation by various strains and species belonging to Bifidobacterium, a genus that includes gut commensals with reported health-promoting activities, is induced by high concentrations of bile (0.5% (w/v) or higher) and individual bile salts (20 mM or higher), rather than by acid or osmotic stress. The transcriptomic response of a bifidobacterial prototype Bifidobacterium breve UCC2003 to such high bile concentrations was investigated and a random transposon bank of B. breve UCC2003 was screened for mutants that affect biofilm formation in order to identify genes involved in this adaptive process. Eleven mutants affected in their ability to form a biofilm were identified, while biofilm formation capacity of an insertional mutation in luxS and an exopolysaccharide (EPS) negative B. breve UCC2003 was also studied. Reduced capacity to form biofilm also caused reduced viability when exposed to porcine bile. We propose that bifidobacterial biofilm formation is an adaptive response to high concentrations of bile in order to avoid bactericidal effects of high bile concentrations in the gastrointestinal environment. Biofilm formation appears to be a multi-factorial process involving EPS production, proteins and extracellular DNA release, representing a crucial strategy in response to bile stress in order to enhance fitness in the gut environment.