Cyclic lipopeptides and particularly surfactins produced by Bacillus species retain antibacterial, antiviral, biofilm-forming and plant resistance-inducing activities. In most cases, their role in biological control of plant diseases was evoked on the basis of in vitro assays or by using non-producing/overproducing mutants but there is a need for more direct evidence of an efficient lipopeptide biosynthesis in the rhizosphere. In this work, we coupled LC-MS quantification of the lipopeptides secreted by cells colonizing tomato plants with the use of psrfA-lacZ reporter system integrated within the BGS3 chromosome to study the expression of the surfactin operon in planta. Results showed that a higher level of psrfA induction was observed upon the establishment of a stable BGS3 population on roots and surfactins extracted from the rhizosphere were produced in biologically significant quantities. Our results also demonstrate that BGS3 efficiently utilizes the main substrates from plant exudates to produce surfactins. This synthesis is also efficient in cells forming colonies and the production may be favoured in bacteria developing slowly in the rhizosphere. This provides a first understanding of how environmental factors may influence lipopeptide production by beneficial Bacillus strains.