Here, the purification, biochemical and biological properties of a trypsin inhibitor from Inga vera seeds (IVTI) are described. Partial amino acid sequence of IVTI showed that it belongs to the MEROPS I03 Kunitz inhibitor family. Moreover, it is composed of a single 20 kDa polypeptide chain with one disulfide bridge and was capable of inhibiting bovine trypsin at a 1:1 molar ratio with a 1.15 nM inhibition constant. IVTI was stable over a wide range of temperature, pH and concentration of a reducing agent. IVTI also inhibited the trypsin-like enzymes from the midgut of lepidopteran pests, such as Anagasta kuehniella (89%), Spodoptera frugiperda (83%), Corcyra cephalonica (80%), Heliothis virescens (70%) and Helicoverpa zea (64%). Furthermore, bioinsecticidal assays against A. kuehniella demonstrated that IVTI affected larval development by impairing weight gain and survival, as well as altering the duration of the larval cycle. IVTI was also fungicidal to Candida buinensis and bacteriostatic agent to Escherichia coli. Further assays revealed that IVTI is a chemopreventive agent against human epithelial colorectal adenocarcinoma cells (CACO-2), reducing cell viability by 70% at 200 μg.mL−1. In summary, these results demonstrate the multifaceted potential of IVTI as a biotechnological tool for agriculture and healthcare.