Exogenous fatty acids (exoFAs) have been reported to give bacteria such as E. faecalis and S. aureus an advantage over certain membrane-targeting antibiotics, like daptomycin. The impact of exoFAs on conferred tolerance has been most deeply characterized by microbiology assays along with some lipidomic analyses. However, there are key gaps in the understanding of how strains with different genetic determinants for resistance utilize structurally similar FAs and the roles of certain enzymes in exoFA usage or de novo FA synthesis. To begin to address this first gap, the impact of FA 18:1(9z), which is known to provide protection to daptomycin, was examined in daptomycin-susceptible and -resistant E. faecalis strain pairs using growth and survival assays and LC-IM-MS based lipidomics. Since it is difficult to distinguish exogenous from endogenous FA 18:1 with standard lipidomics, OzID C=C location and stable isotope labeling strategies were utilized to differentiate the OA from endogenous FA 18:1(11z). To address the role of different enzymes involved in exoFA utilization, S. aureus strains with genetic knockouts of enzymes related to the fatty acid kinase (fak) pathway, the process responsible for the incorporation of exoFAs into membrane lipids, were examined for their role in de novo FA synthesis and preference for certain FAs using standard lipidomics. Cumulatively, these analyses add to the ever-growing understanding of how host FAs may be utilized by Gram-positive bacteria to their own advantage over environmental stressors.