Fusarium oxysporum is a destructive soilborne pathogen that causes vascular wilt and root rot in Cannabis sativa, limiting plant health and productivity under greenhouse production. This study evaluated the antagonistic and growth-promoting effects of plant growth-promoting microorganisms, namely Trichoderma asperellum, Bacillus velezensis, Bacillus amyloliquefaciens, and Microbacterium proteolyticum, against a pathogenic F. oxysporum isolate in vitro and in vivo. Dual-culture assays on Potato Dextrose Agar were used to determine inhibitory activity, while scanning electron microscopy was employed to examine pathogen–antagonist interactions. In greenhouse trials, the cannabis cultivar ‘Bergville’ was pretreated with each microorganism and challenged with F. oxysporum to assess disease incidence, disease severity, AUDPC, infection rates, and plant growth responses over 40 days.All microbial treatments significantly suppressed F. oxysporum growth in vitro (p < 0.001). B. velezensis, B. amyloliquefaciens, and M. proteolyticum showed the strongest inhibition, whereas T. asperellum exhibited slower but clear antagonistic activity characterized by hyphal coiling and mycoparasitism. in vivo, all inoculated plants developed disease, but B. amyloliquefaciens provided the greatest reduction in disease progression, with the lowest AUDPC. T. asperellum and B. velezensis also reduced disease advancement, while M. proteolyticum promoted the greatest plant height despite limited biocontrol efficacy. Bacillus treatments improved leaf number and vegetative vigor, although total root and shoot biomass did not differ significantly among treatments.These findings indicate that B. amyloliquefaciens, B. velezensis, and T. asperellum are promising biological control agents for sustainable management of F. oxysporum in cannabis production.