<jats:title>Abstract</jats:title><jats:p>A CE method is described for the enantioselective analysis of propranolol (Prop) and 4‐hydroxypropranolol (4‐OH‐Prop) in liquid Czapek medium with application in the study of the enantioselective biotransformation of Prop by endophytic fungi. The electrophoretic conditions previously optimized were as follows: an uncoated fused‐silica capillary, 4% w/v carboxymethyl‐β‐CD in 25 mmol/L triethylamine/phosphoric acid (H<jats:sub>3</jats:sub>PO<jats:sub>4</jats:sub>) buffer at pH 9 as running electrolyte and 17 kV of voltage. UV detection was carried out at 208 nm. Liquid–liquid extraction using diethyl ether: ethyl acetate (1:1 v/v) as extractor solvent was employed for sample preparation. The calibration curves were linear over the concentration range of 0.25–10.0 μg/mL for each 4‐OH‐Prop enantiomer and 0.10–10.0 μg/mL for each Prop enantiomer (<jats:italic>r</jats:italic>≥0.995). Within‐day and between‐day relative standard deviations and relative errors for precision and accuracy were lower than 15% for all the enantiomers. Finally, the validated method was used to evaluate Prop biotransformation in its mammalian metabolite 4‐OH‐Prop by some selected endophytic fungi. The screening of five strains of endophytic fungi was performed and all of them could biotransform Prop to some extent. Specifically,<jats:italic>Glomerella cingulata</jats:italic>(VA1) biotransformed 47.8% of (−)‐(<jats:italic>S</jats:italic>)‐Prop to (−)‐(<jats:italic>S</jats:italic>)‐4‐OH‐Prop with no formation of (+)‐(<jats:italic>R</jats:italic>)‐4‐OH‐Prop in 72 h of incubation.