We synthesized 2-phenoxyethanol galactoside (PE-Gal) from 2-phenoxyethanol (PE), in which Escherichia coli β-gal (as E. coli cells) and lactose were added in the reaction mixture for galactosylation. About 40 mM PE-Gal was maximally synthesized from about 80 mM PE at 24 h as about 50 % conversion yield. After purifying PE-Gal, the structure of PE-Gal was identified using LC-MS, ¹H NMR, and ¹³C NMR analyses. In addition, it was observed that the water solubility of PE-Gal was increased by galactosylation of PE. The MICs of PE and PE-Gal against Gram-negative and Gram-positive bacteria were fairly similar with each other (23.3–61.3 mM as the average value). PE-Gal was noticeably less cytotoxic against HACAT cells, in particular a remarkable difference in cell viability was observed at concentrations of 20–60 mM PE or PE-Gal. Finally, we accomplished the synthesis of less toxic PE-Gal, compared with PE, using β-gal-containing E. coli cells without changing in the MICs against microorganisms. In the future, PE-Gal will be applicable as a substitute for PE as a less toxic preservative for the cosmetic, pharmaceutical, and food industries.