Our study aimed to use antagonistic fungi, bacteria and chemical formulation, to practically control Fusarium oxysporum, the causal of seasame root-rot and wilt diseases. Trichoderma harzianum, Bacillus subtilis, Pseudomonas fluorescens and chemical antifungal agent (Bion) were tested to evaluate thier capabilities to inhibit Fusarium oxysporum growth in vitro and to control the disease in greenhouse and field trials. All evaluated treatments in the present study, significantly decreased the diseases incidence in the greenhouse compared to control. T. harzianum treatment scored as the most effective one in control study, followed by B. subtilis and P. fluorescens while Bion gave the lowest effect in this regard. Field experiments at Assiut Governorate, during 2016 and 2017 growing seasons were tried. Results of field experiments were conformed with those obtained from the greenhouse experiment. All treatments increased plant height, number of fruiting branches, number of bearing capsules, seed yield per sesame plant and influenced percentage of oil content of sesame plants positively.