Lyophilized whole cells and the crude enzymatic extract from the fungus Stemphylium lycopersici were successfully immobilized in situ in rigid polyurethane foam, as an alternative biocatalyst for the kinetic resolution of rac-1-phenylethylamine, an interesting building block in the synthesis of pharmaceuticals. The enzymatic activity of immobilized whole cells and the crude enzymatic extract was 2.52 and 5.05 U/g, respectively. The immobilization yield in polyurethane of both forms was at least above 100%, and the process did not change the affinity of the biocatalyst for the substrate. The optima reaction conditions for the kinetic resolution of rac-1-phenylethylamine into acetophenone were: 32.5 °C, 120 rpm by 24 h containing 10 mL (20% m/v) of phosphate buffer (pH 7.5) and 20% biocatalyst mass, resulting in conversions of 45% and 34% using the immobilized lyophilized fungus and crude enzymatic extract, respectively. Stemphylium lycopersici was also subjected to high pressure using CO2. The best conditions provided conversions of 49 (99% ee) and 26.21 (without ee) using the immobilized lyophilized fungus and the immobilized lyophilized crude enzyme extract, respectively, with 20 cycles of reuse and recovery greater than 50% (fungus). Interestingly, the compounds were satisfactorily converted to the corresponding ketones with up to 90% ee for the R-enantiomer. The capacity for conversion of the immobilized lyophilized fungus to different amines: rac-1,2,3,4-tetrahydro-1-naphthylamine, rac-1-phenylpropylamine, and rac-phenylbutylamine was also evaluated. ω-transaminase activity changed significantly depending on the experimental conditions applied, allowing the selection of proper operating conditions for advantageous application of this biocatalyst in transamination reactions. •Stemphylium lycopersici as biocatalyst for kinetic resolution of amines.•The efficiency of polyurethane for the immobilization of biocatalysts.•The fungal whole cell contributes positively for ω-transamination reactions.•The activation process in pressurized CO2 provided a better catalytic performance.