The horseradish peroxidase (HRP)-catalyzed kinetic resolution of racemic, secondary hydroperoxides in the presence of guaiacol is reported. Both the catalytic efficiency and the stereoselectivity of HRP highly depend on the structure of the hydroperoxides. The enzyme selectively recognizes sterically uncumbered (R)-configurated alkyl aryl hydroperoxides, which allows kinetic resolution by means of enantioselective reduction to furnish optically pure (S) hydroperoxides and (R) alcohols. Poor enzyme recognition is observed with hydroperoxides possessing branched aliphatic chains and none at all with tertiary hydroperoxides. This enzyme-catalyzed reduction can be performed on preparative scale to provide optically pure hydroperoxides conveniently. Since HRP is known to be composed of different isoenzymes, the kinetic resolution was investigated with the isolated isoenzymes A and C, which showed that mostly isoenzyme C catalyzes the asymmetric reduction of the racemic hydroperoxides.