A recombinant Haematobia irritans irritans trypsin inhibitor (HiTI - Mw 7030 kDa) phagemid library was constructed and displayed functionally on the tip of the filamentous M13 phage. A combinatorial library of 7.2 x 106 mutants was created with HiTI mutations restricted to the P1 - P3 and P5 positions of the reactive site. This combinatorial library was selected for trypsin-like Pr2 proteases of Metarhizium anisopliae fungus, and 11 HiTI mutants containing the following substitutions: K17G, S18R, D19G, S21A, among 60 sequenced clones, were obtained. In order to confirm the inhibitory activity of the selected sequences, we transferred the selected sequence to the shortest protease inhibitor, the sunflower trypsin inhibitor (SFTI - Mw 1533 Da), for inhibitory activity analysis. The hybrid peptide containing the mutated sequence (SFTI-Mut, GRCTRGRGLACFPD-NH2; Ki = 14 μM) presented an apparent inhibition constant (Kiapp) for Pr2 proteases ~20-fold lower than the control peptide containing the original HiTI sequence (SFTIHiTI, GRCTRKSDLSCFPD-NH2; Ki = 259 μM). In conclusion, the present work enabled the selection of a specific HiTI mutant for Pr2 proteases of M. anisopliae fungus using a HiTI combinatorial library on M13 phage surface. Selection of strong binders by phage display and their validation as inhibitors using synthetic hybrid peptides proved to be a powerful technique to generate specific serine protease inhibitors suitable for studies of drug design and enzyme-inhibitor interaction.