Submillimolar concentrations of cytotoxic bile acids (BAs) induce cell death via apoptosis. On the other hand, several cytoprotective BAs acids were shown to prevent apoptosis in the same concentration range. Still, the mechanisms by which BAs trigger these opposite signaling effects remain unclear. This study was aimed to determine if cytotoxic and cytoprotective BAs, at physiologically active concentrations, are able to modulate the biophysical properties of lipid membranes, potentially translating into changes in the apoptotic threshold of cells. Binding of BAs to membranes was assessed through the variation of fluorescence parameters of suitable derivatized BAs. These derivatives partitioned with higher affinity to liquid disordered than to the cholesterol-enriched liquid ordered domains. Unlabeled BAs were also shown to have a superficial location upon interaction with the lipid membrane. Additionally, the interaction of cytotoxic BAs with membranes resulted in membrane expansion, as concluded from FRET data. Moreover, it was shown that cytotoxic BAs were able to significantly disrupt the ordering of the membrane by cholesterol at physiologically active concentrations of the BA, an effect not associated with cholesterol removal. On the other hand, cytoprotective bile acids had no effect on membrane properties. It was concluded that, given the observed effects on membrane rigidity, the apoptotic activity of cytotoxic BAs could be potentially associated with changes in plasma membrane organization (e.g. modulation of lipid domains) or with an increase in mitochondrial membrane affinity for apoptotic proteins.