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ABSTRACT Enterococcus bacteria inhabit human and soil environments that show a wide range of pH values. Strains include commensals as well as antibiotic-resistant pathogens. We investigated the adaptation to pH stress in E. faecalis OG1RF by conducting experimental evolution under acidic (pH 4.8), neutral pH (pH 7.0), and basic (pH 9.0) conditions. A serial planktonic culture was performed for 500 generations and in a high-pH biofilm culture for 4 serial bead transfers. Nearly all of the mutations led to nonsynonomous codons, indicating adaptive selection. All of the acid-adapted clones from the planktonic culture showed a mutation in fusA (encoding elongation factor G). The acid-adapted fusA mutants had a trade-off of decreased resistance to fusidic acid (fusidate). All of the base-adapted clones from the planktonic cultures as well as some from the biofilm-adapted cultures showed mutations that affected the Pst phosphate ABC transporter ( pstA , pstB , pstB2 , pstC ) and pyrR (pyrimidine biosynthesis regulator/uracil phosphoribosyltransferase). The biofilm cultures produced small-size colonies on brain heart infusion agar. These variants each contained a single mutation in pstB2 , pstC , or pyrR . The pst and pyrR mutants outgrew the ancestral strain at pH 9.2, with a trade-off of lower growth at pH 4.8. Additional genes that had a mutation in multiple clones that evolved at high pH (but not at low pH) include opp1BCDF (oligopeptide ABC transporter), ccpA (catabolite control protein A), and ftsZ (septation protein). Overall, the experimental evolution of E. faecalis showed a strong pH dependence, favoring the fusidate-sensitive elongation factor G modification at low pH and the loss of phosphate transport genes at high pH. IMPORTANCE E. faecalis bacteria are found in dental biofilms, where they experience low pH as a result of fermentative metabolism. Thus, the effect of pH on antibiotic resistance has clinical importance. The loss of fusidate resistance is notable for OG1RF strains in which fusidate resistance is assumed to be a stable genetic marker. In endodontal infections, enterococci can resist calcium hydroxide therapy that generates extremely high pH values. In other environments, such as the soil and plant rhizosphere, enterococci experience acidification that is associated with climate change. Thus, the pH modulation of natural selection in enterococci is important for human health as well as for understanding soil environments.