Fungal pathogens induce a variety of diseases both in plants and post-harvest food crops, resulting in significant crop losses for the agricultural industry. Although the usage of chemical-based fungicides is the most common way of controlling these diseases, they damage the environment, have the potential to harm human and animal life, and may lead to resistant fungal strains. As such, there is an urgent need for diverse and effective agricultural fungicides that are environmentally- and eco-friendly. In plants, polygalacturonase-inhibiting proteins (PGIPs) play critical roles for resistance to fungal disease by inhibiting the pectin-depolymerizing activity of endopolygalacturonases (PGs), one type of enzyme secreted by pathogens that compromises plant cell walls and leaves the plant susceptible to disease. Here, the interactions between PGIPs from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) and Glycine max (GmPGIP3), and PGs from Aspergillus niger (AnPG2), Botrytis cinerea (BcPG1, BcPG2), and Fusarium moniliforme (FmPG3) were reconstituted through a yeast two hybrid (Y2H) system to investigate the inhibition efficiency of various PvPGIP1 and 2 truncations and mutants. Investigation on the sequence-function relationships of PvPGIP utilizing a combination of site directed mutagenesis and a variety of peptide truncations suggests that LRR5 could have the most essential structural feature for the inhibitory activities. This suggests it may be a possible target for the future engineering of PGIP with enhanced activity. We found that tPvPGIP2_5-8 and tGmPGIP3_5-8, which contains LRR5 to LRR8 and is only one-third the size of the full-length peptide, exhibits the same level of interactions with AnPG and BcPGs as the full-length PvPGIP2 and GmPGIP3 via Y2H. On pectin assays, application of both full length PvPGIP2 and tPvPGIP2_5-8 secreting yeast or purified PGIP proteins clearly slows down the growth of A. niger and B. cinerea. On plant assays utilizing detached leaves from N. benthamiana, application of both full length and tPvPGIP2_5-8 secreting yeast or purified proteins reduced the growth and infection rate of B. cinerea. Additionally, PvPGIP2 remains thermostable up to 42°C and retains its inhibitory activity against B. cinerea on pectin assays and in planta assays, delaying growth of the pathogenic fungi by up to one week.