This study aimed to evaluate the bio-agent formulations of Trichoderma harzianum, Bacillus subtilis and Pseudomonas aeruginosa for the protection of lupine plants against wilt disease caused by Fusarium oxysporum f. sp. lupini. T. harzianum isolate (T1), P. aeruginosa isolate (P1) and B. subtilis isolate (B3) were the most effective ones in reducing the growth of F. oxysporum f. sp. lupini in vitro. Consequently, these isolates were used as bio-agent to control wilt on lupine plants in greenhouse experiments. The bio-agent isolates administered in the form of cells in broth media, powdered cells, and immobilized cells on sawdust as formulations were tested for their abilities to control lupine wilt in a greenhouse compared to Captan fungicide. Soil treatments with the three bio-agent formulations (broth medium or cells powder and sawdust) showed a markedly great reduction in wilt disease severity. Although all the formulations reduced the wilt disease severity compared to the water treatment, using the cells powder formulation of bio-agent reduced the wilt severity more than broth medium and sawdust formulations. In all formulations, T. harzianum (T1) exhibited the highest effect in the control of wilt compared with other bio-agent treatments. The sawdust and powder bio-agent formulations significantly reduced the disease severity of wilt on Lupine, as well as improved growth parameters (shoot, root, and plant length and seed yield/plant) of lupine plants compared with control treatment.