The sophorolipid class of biosurfactants is finding increasing use in personal care as well as pharmaceutical products and has the potential to disrupt biofilm formation and inhibit the growth of a variety of clinically relevant organisms. In order to investigate potential biomedical applications of sophorolipids derived from nonpathogenic organisms, we fractionated and purified glycolipid biosurfactant sophorolipids produced by the yeast Starmerella bombicola, which yielded nonacetylated acidic C18:1 congeners that were essentially free from other contaminants (>95% purity). These acidic sophorolipids have antimicrobial activities against the nosocomial infective agents Enterococcus faecalis and Pseudomonas aeruginosa, with significant reductions in CFU at concentrations of as low as 5 mg ml−1. In addition, the sophorolipid showed similar effects against the same two bacterial strains when combined with kanamycin or cefotaxime. As a potential use of these sophorolipids is as a component of topically applied creams for the treatment of wound infections, it is clear that they must have no demonstrable adverse effect on wound healing. To assess this, we evaluated mammalian cell toxicity in vitro using viability tests, which revealed no adverse effect on either endothelial or keratinocyte-derived cell lines with sophorolipid concentrations of < 0.5 mg ml−1. In addition, in vivo experiments using a mouse skin wounding assay revealed that the time course of healing wounds was unaffected by the application of sophorolipid-containing creams, and histological examination of regenerated skin tissue confirmed that the healing process was similar to that observed for control animals, with no evidence of inflammation. These results are consistent with the suggestion that acidic sophorolipids can be used as a component of antimicrobial creams to reduce the risk of wound infection during healing.