Trichoderma harzianum and Aneurinobacillus migulanus were tested separately and in combination as potential biological control agents against F. oxysporum f. sp. gladioli on gladiolus in soil-less culture.  The major secondary metabolites produced by Trichoderma harzianum against F. oxysporum f. sp. gladioli in the treated gladiolus corms as well as in the potato dextrose broth were studied.  The impact of re-application times on the effectiveness of Trichoderma harzianum and Aneurinobacillus migulanus against gladiolus corm rot and Gladiolus production in soil-less culture was examined. Results showed that T. harzianum proved to use different mechanisms against F. oxysporum f. sp. gladioli depending on  the inoculation method. T. harzianum and A. migulanus reduced host defence enzymes.  Also, they increased GA ₃ and IAA content in the corms.  SEM showed that the suppressive mechanisms of T. harzianum and A. migulanus differed T. harzianum appeared to operate through a combination of antibiosis and substrate competition, 6-n-pentyl-6H-pyran-2-one (6PP) and harzianic acid (HA) were found as the main metabolites secreted by T. harzianum.  A significant positive correlation was revealed between qPCR and CFU for both T. harzianum and F. oxysporum f. sp. gladioli.  Multiple applications of T. harzianum and A. migulanus re- application was a successful strategy for suppression of F. oxysporum f. sp. gladioli.  Briefly, T. harzianum was more effective than A. migulanus in F. oxysporum f. sp. gladioli suppression and Gladiolus growth enhancement in soil-less culture. The mixing strategy between T. harzianum and A. migulanus failed.