Effective skin antisepsis is of central importance in the prevention of wound infections, colonization of medical devices, and nosocomial transmission of microorganisms. Current antiseptics have a suboptimal efficacy resulting in substantial infectious morbidity, mortality, and increased health care costs. Here, we introduce an in vitro method for antiseptic testing and a novel alcohol-based antiseptic containing 4 to 5% of the polar aprotic solvent dimethyl sulfoxide (DMSO). The DMSO-containing antiseptic resulted in a 1- to 2-log enhanced killing of Staphylococcus epidermidis and other microbes in vitro compared to the same antiseptic without DMSO. In a prospective clinical validation, blood culture contamination rates were reduced from 3.04% for 70% isopropanol-1% iodine (control antiseptic) to 1.04% for 70% isopropanol-1% iodine-5% DMSO (P < 0.01). Our results predict that improved skin antisepsis is possible using new formulations of antiseptics containing strongly polarized but nonionizing (polar aprotic) solvents.