The work describes a novel supercritical fluid chromatography method for the separation of four stereoisomers, RZ(+), SZ(-), RE(+) and SE(-) of luliconazole, an antifungal agent on amylose tris[(S)-α-methylbenzyl carbamate] based Chiralpak IH column. The effect of organic modifiers (methanol, ethanol and isopropanol), column temperature and back pressure were evaluated for their selective separation. A consistent elution order, RZ(+) > SZ(-) > RE(+) > SE(-) was observed in all the modifiers. Amongst the three modifiers, the best result in terms of selectivity, resolution and analysis time was obtained with isopropanol. Analytical separation (Rs > 1.5) of RZ(+) and SZ(-) & RE(+) and SE(-) pairs was achieved with a mobile phase consisting of CO2: isopropanol (80: 20, v/v) within 5.0 min. The retention of isomers increased with increase in temperature and decreased with increase in pressure, which was more prominent for RE(+) and SE(-) isomers. The van't Hoff plots revealed that the chiral separation process was essentially entropy driven. Molecular docking was performed to understand the type of chiral recognition between the stereoisomers and the chiral stationary phase and to understand their elution orders under optimized conditions. The results suggested hydrogen bonding and π–π interactions, as the dominant interaction modes. The elution order and binding energy of the interactions were in good agreement with the experimental results. Quantitative studies of RE(+) luliconazole the pharmacologically active isomer was also performed using a marketed formulation.