High-performance liquid chromatography (HPLC) was used for the enantiomeric separation of chiral imidazole derivatives endowed with antimycotic activity. Enantioselective columns, containing carbamates of cellulose and amylose, were used. The influence of the nature and content of an alcoholic modifier in the mobile phase was studied. The isolated enantiomers, separated on semipreparative columns, were submitted to in vitro biological investigations.