Fungal biotransformations have become very important in the study of chiral drugs because the reactions performed by these microorganisms may be enantioselective. However, analyses of analytes present in liquid culture medium have proved to be very difficult due to the complexity of this matrix. The aim of this work was to couple dispersive liquid-liquid microextraction (DLLME) with capillary electrophoresis to evaluate the biotransformation of the antidepressant drug venlafaxine (Vx) into its chiral metabolites, N-desmethylvenlafaxine (NDV) and O-desmethylvenlafaxine (ODV) by fungi. The chiral separation was carried out in 50 mmol L-1 sodium phosphate buffer pH 2.0 containing 8 mmol L-1 α-cyclodextrin and 1.0% (m/v) carboxymethyl-β-cyclodextrin. The temperature of the capillary was set at 20 °C. A voltage of +20 kV was applied during analysis. The DLLME was accomplished using 300 µL of isopropanol (disperser solvent) and 200 µL of chloroform (extraction solvent). The method was completely validated and showed to be linear over the concentration range of 75-938 ng μL-1 for ODV and NDV enantiomers and of 500-15000 ng μL-1for venlafaxine enantiomers with a correlation coefficient higher than 0.99. The selectivity of the method was evaluated and no interference peaks were detected in the migration time of the analytes. The limit of quantification was 75 ng μL-1 for metabolite enantiomers and 500 ng μL-1 for venlafaxine enantiomers. The study showed a stereoselective biotransformation of venlafaxine into (+)-(S)-N-desmethylvenlafaxine by the fungus Cunninghamella elegans ATCC 10028B with an enantiomeric excess of 100%.