Chiral 2-hydroxypropanone derivatives 5a−v, 8a−d, and 10a, b were formed by benzoylformate decarboxylase (BFD) catalyzed C−C bond formation. A donor aldehyde and acetaldehyde as an acceptor were carboligated in aqueous buffer solution with remarkable ease in high chemical yield and good to high optical purity. The substrate range of this thiamin diphosphate dependent enzyme was examined to employ this benzoin condensation type reaction in stereoselective synthesis. The observed dependence of the enantiomeric excess on the substitution pattern could be exploited to design substrates resulting in high selectivity. Best substrates with regard to optical purity were meta-substituted benzaldehyde derivatives. To enable a general and convenient applicability of the BFD-catalyzed C−C bond formation, analytical batch experiments were scaled up to give (S)-2-hydroxy ketones in good to high yields on a preparative scale. Further, the solubility of some of the organic substrates in aqueous solution was increased by the use of cyclodextrin or buffer/DMSO mixtures.