(R)-2-Chloromandelic acid (R-CM) is one of the chiral building blocks used in the pharmaceutical industry. As a result of screening for microorganisms that asymmetrically hydrolyze racemic 2-chloromandelic acid methyl ester (CMM), Exophiala dermatitidis NBRC6857 was found to produce R-CM at optical purity of 97% ee. The esterase that produces R-CM, EstE, was purified from E. dermatitidis NBRC6857, and the optimal temperature and pH of EstE were 30°C and 7.0, respectively. The estE gene that encodes EstE was isolated and overexpressed in Escherichia coli JM109. The activity of recombinant E. coli JM109 cells overexpressing estE was 553 times higher than that of E. dermatitidis NBRC6857. R-CM was produced at conversion rate of 49% and at optical purity of 97% ee from 10% CMM with 0.45 mg-dry-cell/L recombinant E. coli JM109 cells. Based on these findings, R-CM production by bioconversion of CMM may be of interest for future industrial applications.