Amphotericin B (AmB) is a commonly used antifungal drug, with well-documented effects on cellular immune responses. We determined that AmB-stimulated γδ T-cell activation and proliferation in vitro at very low concentrations. AmB also enhanced IFN-γ production by NK cells in combination with IL-18. AmB had a greater effect on IFN-γ production in cells isolated from very young animals. Although innate immunostimulatory aspects of AmB have been defined, AmB has not been extensively applied in non-fungal infection settings. Given that γδ T cells are increased and activated in Salmonella infection in cattle, we assessed the effects of AmB in protection from Salmonella enterocolitis in calves. One injection of AmB, at approximately one-tenth of the concentration used in human patients to counter fungal infection, or saline control, was delivered intravenously to calves prior to infection with Salmonella. This single injection caused no adverse effects, reduced disease symptoms from Salmonella enterocolitis and significantly reduced Salmonella bacteria shed in feces of infected animals. Our findings suggest that AmB may be an inexpensive and readily available prophylactic approach for the prevention of bacterial infection in calves.