Small natural or synthetic peptides have been reported to exhibit potent inhibitory capability against trypsin, some of which were also found to have antibacterial potency. Here, we described a successful application of in silico-in vitro integrated approach to rationally design and optimize bifunctional peptides with both trypsin inhibitory and antimicrobial activities. In the procedure, computer-aided methods including protein docking, peptide redocking, molecular dynamics simulations and binding free energy calculations were employed to model and analyze the intermolecular interaction between human trypsin (hT) and natural trypsin inhibitors (TIs). Based on the modeled hT–TI complex structures a number of promising peptide fragments were derived from the trypsin inhibitory loop of TIs, which were then tested experimentally to determine their inhibitory potency on recombinant hT protein as well as their antibacterial potency against three clinical strains. Consequently, few peptides were found to possess a good profile of trypsin inhibitory and antibacterial bi-functionality. Structural visualization and noncovalent examination of hT complex with a potent peptide revealed that the hydrophobic forces and van der Waals contacts between the peptide nonpolar residues and the hydrophobic pocket around hT active site confer significant stability to the complex architecture, while few specific hydrogen bonds and cation-π interactions at the complex interface contribute to peptide selectivity for hT.