A Bacillus subtilis strain, isolated from topsoil by a selective screening procedure, is effective for the biocatalytic kinetic resolution of racemic hydroperoxides: A variety of secondary aralkyl hydroperoxides have been resolved by this microorganism in moderate to high enantioselectivities [up to 88% ee for the (R)-hydroperoxide]. This topsoil bacterium B. subtilis also reduced the racemic tertiary 1-methyl-(1-phenyl)propyl hydroperoxide enantioselectively to afford the (R)-hydroperoxide in 39% ee. In contrast to the bacteria, the fungus Aspergillus niger displayed the reverse sense of enantioselectivity in the kinetic resolution of the racemic hydroperoxides, that is, the (R) enantiomer was preferably reduced and the (S)-hydroperoxide was obtained in enantiomerically enriched form. Such an inverse enantioselectivity has not been observed before for the peroxidases of bacterial versus fungal and plant sources. Furthermore, it was shown that immobilized B. subtilis cells may be reused for several catalytic cycles in the kinetic resolution of racemic hydroperoxides without any loss of enzymatic activity. This microbial process offers a promising biocatalytic methodology for the preparation of optically active hydroperoxides.