Phytopathogenic fungi devastate agricultural crops worldwide. The biological agents, such as Trichoderma spp., antagonize phytopathogenic fungi by secreting various cell wall‐degrading enzymes, for example, endochitinase and β‐1,3‐glucanase that target glycosidic linkages in β‐glucan and chitin polymers of fungal cell walls, thus inhibiting pathogen growth. In this study, two antifungal genes endochitinase and β‐1,3‐glucanase cloned from local Trichoderma spp. were ligated in pET28a+ expression vector individually to generate two recombinant vectors. The vectors were mobilized into Escherichia coli host strain Rosetta‐gami 2 for protein expression, and the 6xHis‐tagged recombinant proteins were purified through Ni‐NTA affinity chromatography. The purified proteins were individually confronted in vitro with pure cultures of Phytophthora parasitica (destructive pathogen affecting several hundred plant species worldwide) for analyzing their effect on pathogen growth. In vitro confrontation assay revealed P. parasitica growth inhibition by purified β‐1,3‐glucanase. The pathogen growth inhibition was due to hyphal morphological distortions, such as breakages, swelling, and holes evinced through electron micrography confirming direct role of β‐1,3‐glucanase in pathogen structural degradation.