To identify potential applications of nineteen esterases from thermophiles, we mapped their substrate selectivity and enantioselectivity using a library of 50 esters. We measured the selectivities colorimetrically using Quick E, which uses pH indicators to detect hydrolysis and a chromogenic reference compound as an internal control. The substrate selectivity mapping revealed one esterase, E018b, with a strong preference for acetyl esters (14- to 25-fold over hexanoate). The enantioselectivity mapping revealed a number of cases of high enantioselectivity. Thirteen of the 19 esterases showed moderate or better enantioselectivity (>19) toward 1-phenethyl butyrate favoring the (R)-enantiomer and two esterases (E008, E013) showed moderate or better enantioselectivity (>20) toward methyl 2-chloropropionate favoring the (S)-enantiomer. Three esterases (E001, E004, E005) showed high (>46) enantioselectivity toward menthyl acetate favoring the (R)-enantiomer. This rapid mapping of the selectivity simplifies the characterization of new enzymes.