Antimicrobial lipids on skin are proposed to form a barrier against microbial colonisation. Skin lipids, such as unsaturated fatty acids and sphingosines, cause membrane permeabilisation and/or proton motive force disruption . These lipids may be crucial in determining the diversity and degree of staphylococcal skin colonisation. Specifically, antimicrobial lipids may inhibit skin colonisation by Staphylococcus aureus while permitting the growth of Staphylococcus epidermidis. Here it was shown that skin fatty acids sapienic acid and linoleic acid are more active against S. aureus than S. epidermidis. This supports a role for fatty acids in the prevention of S. aureus skin colonisation. The most anti-staphylococcal skin lipid tested was D-sphingosine; no differences in resistance levels between S. aureus and S. epidermidis to D-sphingosine were observed. The genetic response and basis for resistance to skin antimicrobial lipids of S. epidermidis and S. aureus was investigated using next generation sequencing. The transcriptomic response of both species to sapienic acid was determined using RNA-Seq. Additionally, S. epidermidis and S. aureus were passaged in sapienic acid or D-sphingosine. Isolates with increased lipid resistance after passaging were genome sequenced, and mutations associated with increased resistance were characterised. From these approaches, several genes and pathways potentially involved in the responses of both species to skin lipids became apparent. These components included cell wall biosynthesis, transport and production of small molecules, ammonia production, albumin binding proteins and putative lipid efflux pumps. Cellular components identified as specifically involved in S. aureus resistance to sapienic acid included capsule and staphyloxanthin biosynthesis. Cellular components involved specifically in S. epidermidis resistance to sapienic acid were also speculatively identified, though the functions of these components were not resolved. This study has increased our understanding of staphylococcal molecular interactions with host antimicrobial lipids, which could lead to applications in the design of novel antimicrobial compounds.