Teixobactin is a new antimicrobial active against a number of multidrug-resistant pathogens, including Staphylococcus aureus and Enterococcus faecalis, with no reported mechanisms of teixobactin resistance. Understanding how resistance could develop will be crucial to the success and longevity of teixobactin as a new potent antimicrobial. Antimicrobial tolerance has been shown to facilitate the development of resistance and we show E. faecalis is intrinsically tolerant to teixobactin at high concentrations. We subsequently chose E. faecalis as a model to elucidate the molecular mechanism underpinning teixobactin tolerance and to understand how this may contribute to the development of teixobactin resistance. We attempted to isolate teixobactin resistant and/or tolerant E. faecalis mutants using transposon mutagenesis and spontaneous mutagenesis via long-term exposure and serial passaging. No mutants displaying altered susceptibility to teixobactin were isolated using transposon mutagenesis and long-term exposure. Six mutants were isolated from three strains of E. faecalis by spontaneous mutagenesis using serial passaging resulting in both higher minimum inhibitory concentration (MIC) values of 4 mg/L (2-fold increase over wild-type) and minimum bactericidal concentration (MBC) values of 64 - >128 mg/L (2-8-fold increase over wild-type). Whole-genome sequencing of these mutants identified mutations in cell wall biosynthesis, with isoprenoid biosynthesis (mvaD/E) and exopolysaccharide transport (epaM) appearing to play important roles. Further analysis showed that mutants displayed a reduced cell wall thickness with significant changes in gene expression in the absence of teixobactin compared to the parental strain exposed to teixobactin-induced antimicrobial stress. We propose that mutations in mvaD/E and epaM results in a decrease of cell wall biosynthesis and activation of an “antimicrobial stressed” state.