Chiral separation of 12 new triadimenol antifungal active compounds by electrokinetic chromatography and chiral recognition mechanisms by computer-aided molecular modeling techniques were studied. Seven neutral cyclodextrins were used as chiral selectors. Heptakis-(2,3,6-tri-O-methyl)--cyclodextrin (TM--CD) exhibited a very high enantioselectivity power to 12 active compounds compared to the other tested CDs. The influences of the concentration of TM--CD, buffer pH, buffer concentration, applied voltage, and temperature were investigated, respectively. Under the optimum separation conditions, all the 12 active compounds were baseline separated and the resolutions of most compounds were beyond 2.50. The study of the analyte structure-enantioseparation relationships showed that substitutions in the side chains played important roles on enantiomeric separation. By means of computer-aided molecular modeling software Discovery Studio 2.5/Sybyl 7.0/Gold 3.0.1, inclusion process between TM--CD and these enantiomers was investigated and their binding energies were calculated. The results suggested that the enantioseparation result related to the difference in binding energy. And the good separation obtained in the presence of the TM--CD chiral selector was due to the big binding energy difference of two enantiomers with the chiral selector.