The emergence of antibiotic resistance poses a serious and challenging threat to healthcare systems, making it imperative to discover novel therapeutic options. This work reports the isolation and characterization of a thermostable trypsin inhibitor from chia (Salvia hispanica L.) seeds, with antibacterial activity against Staphylococcus aureus sensitive and resistant to methicillin. The trypsin inhibitor ShTI was puri ed from chia seeds through crude extract heat treatment, followed by a nity and reversed-phase chromatography. Tricine-SDS-PAGE revealed a single glycoprotein band of ~11 kDa under nonreducing conditions, con rmed by mass spectrometry analysis (11.558 kDa). ShTI was remarkably stable under high temperatures (100 °C; 120 min.) and a broad pH range (2 -10; 30 min.).Upon exposure to DTT (0.1 M; 120 min.), ShTI antitrypsin activity was partially lost (~ 38%), indicating the participation of disul de bridges in its structure. ShTI is a competitive inhibitor (Ki = 1.79 x 10-8 M; IC50 = 1.74 x 10-8 M) that forms a 1:1 stoichiometry ratio for the ShTI: trypsin complex. ShTI displayed antibacterial activity alone (MICs range from 15.83 to 19.03 µM) and in drug combination with oxacillin (FICI range from 0.20 to 0.33) against strains of S. aureus, including methicillin-resistant strains.Overproduction of reactive oxygen species and plasma membrane pore formation are involved in the antibacterial action mode of ShTI. Overall, ShTI represents a novel candidate for use as a therapeutic agent for the bacterial management of S. aureus infections.