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Enterobacter cloacae, Microbacterium oleovorans, Pseudomonas solanacearum and Bacillus subtilis were investigated in order to evaluate: (i) the inoculum size of two bacterial mixtures on Fusarium verticillioides growth and fumonisin B1 production in vitro at different water activities and (ii) the efficacy of a seed treatment with the best bacterial mixture on F. verticillioides root colonization in greenhouse studies.The influence of bacterial mixtures (1 = E. cloacae and M. oleovorans and 2 = P. solanacearum and B. subtilis) to antagonize 13 F. verticillioides strains at different inoculum concentrations (108, 109 and 1010 cells ml−1) and water activities (0·937, 0·955 and 0·982 aW) were examined. Antibiosis, growth rate and fumonisin B1 production were determined. Bacterial mixture 1 proved to exert the most effective control. Seed treatment with mixture 1 at 108 cells ml−1 had the best inhibitory effect on F. verticillioides root colonization.These results suggest that the combination E. cloacae and M. oleovorans has the potential for the biological control of F. verticillioides as a maize seed inoculant.The application of this knowledge contributes to prevent the vertical transmission of F. verticillioides.