Over the past decade, researchers have tried to decipher the borderline between serine proteases and carboxylic ester hydrolases; although they share the same catalytic triad, the factor that distinguishes their substrate preference still remains unknown. So far, the total inversion of the catalytic character has not yet been fulfilled; only minor improvements of the promiscuous amidase activity have been achieved for the carboxylic ester hydrolases compared to their natural activities, even though different sophisticated approaches of rational design have been applied. In the present study we used a combination of directed evolution and rational design to improve the promiscuous amidase activity of a Bacillus subtilis esterase. A double mutant was identified with 7.5 fold higher relative reaction specificity for the promiscuous amidase reaction with both aromatic and aliphatic substrates, whereas the esterase activity was not significantly affected. The mutation that resulted from the directed evolution experiments was not in the direct vicinity of the active site but affected the substrate binding through a π–π stacking network.