A number of reports have indicated a relationship between bacterial resistance to antibiotics and their lipid composition. In the present study, we characterized the lipid profiles of American Type Culture Collection (ATCC) and clinical strains of Staphylococcus aureus and its correlation with antibiotic resistance and hydrophobicity. The following strains were used: S. aureus ATCC 6538P, S. aureus ATCC 43300 (MRSA), seven clinical strains from the pharynges, two strains from duodenal ulcers, four strains from hip prostheses, and one strain from the conjunctiva. Lipid-related differentiation was observed across the S. aureus strains: the higher abundance of anteiso-pentadecanoic acid (anteiso-C15:0) and anteiso-heptadecanoic acid (anteiso-C17:0), followed by iso-pentadecanoic acid (iso-C15:0), suggested that these were common lipids. Iso-tridecanoic acid (iso-C13:0) and anteiso-tridecanoic acid (anteiso-C13:0), iso-hexadecanoic acid (iso-C16:0) and anteiso-hexadecanoic acid (anteiso-C16:0), and all forms of octadecanoic acid (C18:0) were usually detected in low abundance. Strains isolated from pharynges showed the highest ratio of branched/straight chains. A distinction in two clusters based on the amount and type of bacterial lipids identified was obtained, which correlated to the antibiotic resistance, the strains origin, and the cell-surface hydrophobicity. We report a potential correlation between the lipid profile of S. aureus strains, site of infection, antibiotic resistance, and cell-surface hydrophobicity. These results, which still need further insights, could be a first step to identifying antibiotic resistance in response to environmental adaptation.