Fungicides are very important and diverse environmental and agricultural concern species. Their determination in commercial formulations or environmental matrices, requires highly efficient, selective and sensitive methods. A significant number of these chemicals are chiral with the activity residing usually in one of the enantiomers. The different toxicological and degradation behavior observed in many cases for fungicide enantiomers, results in the need to investigate them separately. For this purpose, separation techniques such as GC, HPLC, supercritical fluid chromatography (SFC) and CE have widely been employed although, at present, HPLC still dominates chromatographic chiral analysis of fungicides. This review covers the literature concerning the enantiomeric separation of fungicides usually employed in agriculture grouping the chiral separation methodologies developed for their analysis in environmental, biological, and food samples.