Phytosphingosine (PS) is a natural antimicrobial ingredient present in the mammalian stratum corneum of the skin. The skin surface is the first line defense system against microbial colonization and infections. The aim of this study was to investigate in vitro antimicrobial effects of PS and PS-hydrochloride (PSHCl) nanoemulsion (NE) formulations and solutions against bacteria and fungi. Minimum inhibitory concentrations (MICs) of the PS-NE and PSHCl-NE formulations and PS/PSHCl solutions were determined by microdilution method against Gram-positive/Gram-negative bacteria and yeasts. Inhibitory effects were also examined by disc diffusion method and inhibition zone diameters were determined. PS/PSHCl solutions and PS-NE/PSHCl-NE formulations were prepared in different conditions. Physicochemical properties of the NEs were characterized and antimicrobial activities were evaluated. In this study we reported that the PS and PSHCl and NEs had variable antimicrobial activities for a variety of Gram-positive bacteria (MIC range 1 μg/ml to 1024 μg/ml), Gram-negative bacteria (MIC range 8 μg/ml to 1024 μg/ml) and Candida strains (MIC range 8 μg/ml to 512 μg/ml). Sphingolipids are part of the natural defense system of the body. We demonstrated that PS and PSHCl solutions and NEs have broad antimicrobial activity against bacteria and yeasts. In addition, the newly developed formulations may have potential for prophylactic and therapeutic intervention in infections.