The effect of pressure was studied in an enzymatic reaction with an immobilized biocatalyst. Naringinase immobilized by entrapment in calcium alginate beads was the biocatalyst used to catalyze, at high-pressure, the hydrolysis of naringin to naringenin. These molecules have great potential in the pharmaceutical industry due to their recognized anti-oxidant, anti-inflammatory, anti-carcinogenic, anti-hypertensive and hypocholesterolemic effects. At high-pressure, the influence of relevant parameters on naringinase catalytic activity such as temperature, substrate concentration, and biocatalyst reuse was studied. At 160 MPa, naringinase entrapped in Ca-alginate beads displayed higher activity, namely in the range of 35–40 °C, whereas the optimum, at atmospheric pressure, was 35 °C. The immobilized naringinase presented a Michaelis–Menten kinetic, with a 65% higher maximum initial rate ( V max ap = 0.069 mM mi n − 1 ) , and a 70% lower K M ap ( 0.097 mM ) at 160 MPa, as compared to kinetic parameters, at atmospheric pressure ( V max ap = 0.042 mM min − 1 and K M ap = 0.303 mM ) . A positive effect of pressure on naringin hydrolysis by immobilized naringinase in Ca-alginate beads was confirmed with a negative activation volume (Δ V ≠) of −9 mL mol −1. The stability of immobilized naringinase was also evaluated at high-pressure.