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Sphingosine is known as a natural antimicrobial agent, protecting the human skin from bacterial colonization and possibly affecting the intestinal microbial community after ingestion. In this study we further investigated the antibacterial spectrum of dietary d-eythro-sphingosine in saline towards three intestinal pathogens and to the health promoting lactobacilli and bifidobacteria. The degree of bactericidal effect was studied using plate counts and Live/Dead analysis combined with flow cytometry. To assess activity under complex intestinal conditions, sphingosine was dosed to the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) for a period of 11 days. Finally, we tried to elucidate the factors influencing the activity and the mode of action of sphingosine. In all performed experiments, high correlation occurred between plate counts and Live/Dead analysis. In saline a strong antibacterial effect was seen to all tested species, Gram-negative and Gram-positive, and sphingosine not selectively acted against pathogens, as health promoting bacteria were also affected. Under simulated intestinal conditions however, no shifts in bacterial concentrations were detected. Experiments with individual medium components thought that the effect of sphingosine is very easily neutralized by BSA, stearic acid and surfactants. Based on our results, d-erythro-sphingosine would only be active when protonated and its mode of action would imply electrostatic attraction to the bacteria and disruption of membrane integrity. In conclusion, the application of sphingosine is limited to specific environments, as activity was very sensitive to inhibition. Yet, because of its broad spectrum membrane disrupting activity, it could be very useful under controlled conditions.