<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>The consequence of simultaneous and sequential inoculation of<jats:italic>T. asperellum</jats:italic>and<jats:italic>B. amyloliquefaciens</jats:italic>cultures with respect to growth rate, differential expression of vital genes and metabolites were examined.</jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>The competition was observed between<jats:italic>T. asperellum</jats:italic>and<jats:italic>B. amyloliquefaciens</jats:italic>under co-cultivation. The proliferation of<jats:italic>Trichoderma</jats:italic>was reduced in the simultaneous inoculation (TB1) method, possibly due to the fastest growth of<jats:italic>Bacillus</jats:italic>. Both<jats:italic>T. asperellum</jats:italic>and<jats:italic>B. amyloliquefaciens</jats:italic>were proliferated in sequential inoculation method (TB2). The sequential inoculation method (TB2) upregulated the expression of metabolites and vital genes (sporulation, secondary metabolites, mycoparasitism enzymes and antioxidants) in<jats:italic>Trichoderma</jats:italic>and downregulated in<jats:italic>Bacillus</jats:italic>and vice versa in co-inoculation method (TB1). The metabolic changes in the co-culture promoted the maize plant growth and defense potential under normal and biotic stress conditions.</jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>The metabolites produced by the co-culture of<jats:italic>T. asperellum</jats:italic>and<jats:italic>B. amyloliquefaciens</jats:italic>improved the maize plant growth and defense potential under normal and biotic stress conditions.</jats:sec>