Secondary metabolites play a pivotal role in the antagonistic activities of some biocontrol species of Trichoderma sp. T. harzianum used previously as biological control agent against different pathogens. In this work, the effect of F. oxysporum f. sp. gladioli on the major secondary metabolites secreted by biocontrol strain of T. harzianum in Potato Dextrose Broth (PDB) cultures were investigated and quantified; followed by investigating the effect of the previous microorganisms on T. harzianumsecondary metabolites secreted in Gladiolus grandiflorus corms tissues under controlled conditions. Thin Layer Chromatography (TLC), Liquid chromatography Mass spectroscopy (LC/MS), and Nuclear Magnetic Resonance (NMR) techniques were used in this study to determine the major secondary metabolites. This is the first report recording the isolation, characterization and quantification of 6-n-pentyl-6H-pyran-2-one (6PP) and harzianic acid (HA) from the treated corms with T. harzianum. The secreted amounts from both metabolites were increased in the pretreated corms with T. harzianum followed by infection with F. oxysporum f. sp. gladioli. Results provided better understandings of the interaction mechanism between T. harzianum, F. oxysporum f. sp. gladioli which could be used in the future in different gene expression studies and will help in using them as bio fertilizers in biocontrol field.