Bile acids (BAs) play an important role in the digestion of dietary fats and act as signaling molecules. However, due to their solubilizing properties, high concentrations in the gut may negatively affect gut epithelium and possibly afferent fibers innervating the gastrointestinal tract (GI). To determine the effect of BAs on intracellular Ca²⁺ and membrane permeabilization we tested a range of concentrations of two BAs on vagal nodose ganglion (NG) neurons, Chinese Hamster Ovary (CHO), and PC12 cell lines. NG explants from mice were drop-transduced with the genetically encoded Ca²⁺ indicator AAV9-Syn-jGCaMP7s and used to measure Ca²⁺ changes upon application of deoxycholic acid (DCA) and taurocholic acid (TCA). We found that both BAs induced a Ca²⁺ increase in NG neurons in a dose-dependent manner. The DCA-induced Ca²⁺ increase was dependent on intracellular Ca²⁺ stores. NG explants, with an intact peripheral part of the vagus nerve, showed excitation of NG neurons in nerve field recordings upon exposure to DCA. The viability of NG neurons at different BA concentrations was determined, and compared to CHO and PC12 cells lines using propidium iodide labeling, showing threshold concentrations of BA-induced cell death at 400–500 μM. These observations suggest that BAs act as Ca²⁺-inducing signaling molecules in vagal sensory neurons at low concentrations, but induce cell death at higher concentrations, which may occur during inflammatory bowel diseases.