Growing consumer awareness of the potential negative health effects of synthetic antibiotics has prompted the search for more natural preservatives that can improve the safety and quality of food. In this study we report the enzymatic synthesis of N-α-[Carbobenzyloxy]-Ile-Gln (Z-IQ) which is the precursor of Ile-Gln (IQ), a new antibacterial dipeptide, using an aqueous–organic biphasic system formed by 50% (v/v) ethyl acetate in 0.1 M Tris – HCl buffer pH 8. A partially purified proteolytic extract from the fruits ofSolanum granuloso leprosum,named granulosain, proved to be a robust biocatalyst for the synthesis of Z-IQ, eliciting 71 ± 0.10% maximal peptide yield in the above described conditions. After cleaving and purifying IQ dipeptide, antimicrobial activity was assayed againstStaphylococcus aureusATCC 25923,Staphylococcus hominisA17771, andStaphylococcus aureusC00195, and MIC values between 118 ± 0.01 μg/mL and 133.7 ± 0.05 μg/mL were obtained. In addition, IQ showed MIC of 82.4 ± 0.01 μg/mL and 85.0 ± 0.00 μg/mL againstEscherichia coliATCC 25922 andEscherichia coliA17683, respectively. IQ did not show inhibitory activity against single-drug resistance (SDR) strains, such asKlebsiella oxytocaA19438 (SDR) andPseudomonas aeruginosaC00213 (SDR), and against multidrug-resistantEnterococcus faecalisI00125 (MDR). IQ also caused growth inhibition ofHelicobacter pyloriNCTC 11638 and three wild-typeH. pyloristrains, which are sensitive to AML, MTZ, LEV and CLA (H. pylori659), resistant to LEV (H. pylori661 SDR), and resistant to MTZ (H. pylori662 SDR). Finally, this study contributes with a new dipeptide (IQ) that can be used as an antimicrobial agent for food preservation or as a safe ingredient of functional foods.