The persistence of pathogenic Agrobacterium strains as soil-associated saprophytes may cause an inconsistency in the efficacy of the biocontrol inoculants under field condition. The study of the interaction occurring in the rhizosphere between the beneficial and the pathogenic microbes is thus interesting for the development of effective biopesticides for the management of crown gall disease. However, very little is still known about the influence of these complex interactions on the biocontrol determinants of beneficial bacteria, especially Bacillus strains. This study aimed to evaluate the effect of the soil borne pathogen Agrobacterium tumefaciens C58 on root colonization and lipopeptide production by Bacillus velezensis strain 32a during interaction with tomato plants. Results show that the presence of A. tumefaciens C58 positively impacted the root colonization level of the Bacillus strain. However, negative impact on surfactin production was observed in Agrobacterium-treated seedling, compared with control. Further investigation suggests that these modulations are due to a modified tomato root exudate composition during the tripartite interaction. Thus, this work contributes to enhance the knowledge on the impact of interspecies interaction on the ecological fitness of Bacillus cells living in the rhizosphere.