Guava is an important revenue generating crop for small, medium, and commercial guava cultivators all over the world. Nematode infestation is one of the factors that cause declines in fruit production. Researches have proven that the application of plant growth regulators induces the synthesis of defense-related proteins in Guava. IBA is one such plant growth regulator, and its effects on guava plants has not yet been elucidated. Thus, this research is focused on the optimization of IBA concentrations, which results in the induction and production of maximum defense-related proteins to defend against root knot nematode. The present study includes the application of IBA on M. enterolobii-infested experimental guava plants at different concentrations ranging from 100 ppm to 2000 ppm. The synthesis of defense-related proteins is identified with 1000 ppm of IBA. At this concentration, IBA influences the morphological, physiological, and biochemical characteristics and enhances the induction of defense-related proteins in M. enterolobii-infested experimental guava plants. Thus, 1000 ppm of IBA prevents nematode infestation in Lucknow-49 guava plants.