In this study, all stereoisomers of cognac lactone (1) were synthesized with over 99% enantiomeric excess via diastereomeric resolution using N‐benzyloxycarbonyl‐l‐proline (N‐Cbz‐l‐proline) as a resolving agent. N‐Cbz‐l‐proline could be applied for the diastereomeric resolution of both syn‐ and anti‐forms and yielded all diastereomers in over 99% diastereomeric excess. The odour properties of racemic and optically active 1 were evaluated for their orthonasal aromas. All the stereoisomers had different odour characteristics and thresholds. In general, the (3S)‐ and (3R)‐forms had fruity and herbal notes, respectively, and (3R,4S)‐1 had the lowest odour threshold at 0.01 ppm. All stereoisomers, except (3R,4S)‐1, showed antibacterial activity against Staphylococcus aureus. Moreover, (3S,4S)‐1 showed high antibacterial activity against both S aureus and Escherichia coli.