Some plant-beneficial bacteria with biopesticide potential produce antimicrobial compounds that are tightly involved in multitrophic interactions occurring in the phytosphere among which antagonism toward a diverse range of phytopathogens. However, there is a crucial need for an accurate assessment of antibiotic production rate in this environment colonized by these bacteria. In this context, we conducted the present thesis work with the scope to better understand how two different Bacillus genus strains produce lipopeptides in the rhizosphere of plants. In the first part, one of the strains (B. amyloliquefaciens S499) was selected out of other isolates for its technological traits and in vitro inhibition growth activity of plant pathogenic fungi. We further demonstrated its biocontrol potential in tomato open-field experiments where plantings have been devastated by a local fungus preliminary identified as Fusarium semitectum. In a second part, we combined two mass spectrometry-based approaches (electrospray ionization and imaging) to analyze the pattern of surfactin, iturin and fengycin lipopeptide families produced in planta by strain S499. Our results show that rhizosphere conditions are conducive for surfactin synthesis but not for other types of lipopeptides and that the lipopeptide pattern can be markedly influenced by nutritional factors, biofilm formation and oxygen availability. In a last part, surfactin gene expression (srfA) level was evaluated in situ on tomato root using the reporter gene (LacZ) inserted in B. subtilis strain BGS3. Results showed effective expression of srfA and production of surfactin in biologically important level quantities upon establishment of bacterial population on roots. Our results also demonstrate that BGS3 developing in colonies, efficiently utilizes the main substrates from plant exudates to produce surfactins. The production may also be favored in bacteria growing slowly in the rhizosphere. Globally, this work contributes to better appreciate the impact of some environmental factors on the in situ biosynthesis of lipopeptides by strains of Bacillus which is probably an essential step for improving the level and reliability of their efficacy as biological agents for the control of plant diseases.