Acyl transferase from Mycobacterium smegmatis (MsAcT) is a promising biocatalyst because it catalyzes an acyl transfer reaction in aqueous solution, thereby accepting many primary and secondary alcohols as substrates. MsAcT also exhibits high enantioselectivity for a selected number of secondary alcohols. To increase the applicability of this enzyme for the production of optically active compounds, a detailed understanding of the reaction mechanism and the factors that affect enantioselectivity is essential. Herein, quantum chemical calculations are employed to study the reactions of two secondary alcohols, 1-isopropyl propargyl alcohol and 2-hydroxy propanenitrile, for which the enzyme displays opposite enantiopreference, favoring the S enantiomer in the former case and R enantiomer in the latter. A model of the active site has been designed and for both substrates various binding modes are evaluated and the intermediates and transition states along the reaction path are then located. The calculated energy profiles agree with the experimental observations, and reproduce the selectivity outcome. Through a detailed analysis of the geometries of key transition states, insights into the origins of the enantiopreference are obtained.