The commercially available inoculant Bacillus amyloliquefaciens FZB42 is able to considerably reduce lettuce bottom rot caused by Rhizoctonia solani. To understand the interaction between FZB42 and R. solani in the rhizosphere of lettuce we used an axenic system with lettuce bacterized with FZB42, and inoculated with R. solani. Confocal laser scanning microscopy showed that FZB42 could delay the initial establishment of R. solani on the plants. To show which secondary metabolites of FZB42 are produced under these in-situ conditions we developed an UHPLC-qToF-MS based method and identified surfactin, fengycin and bacillomycin D in the lettuce rhizosphere. We hypothesized that lipopeptides and polyketides play a role in enhancing the plant defence responses in addition to the direct antagonistic effect towards R. solani , and used a qRT-PCR based assay for marker genes involved in defence signaling pathways in lettuce. A significant higher expression of PDF 1.2 observed in the bacterized plants in response to subsequent pathogen challenge showed that FZB42 could enhance the lettuce defence response towards the fungal pathogen. To identify if surfactin or other non-ribosomally synthesized secondary metabolites could elicit the observed enhanced defence gene expression, we examined two mutants of FZB42 deficient in production of surfactin and the lipopetides and polyketides by expression analysis and pot experiments. In the absence of surfactin and other non-ribosomally synthesized secondary metabolites there was no enhanced PDF 1.2 mediated response to the pathogen challenge. Pot experiment results showed that the mutants failed to reduce disease incidence in lettuce as compared to the FZB42 wild type, indicating, that surfactin as well as other non-ribosomally synthesized secondary metabolites play a role in the actual disease suppression and on lettuce health. In conclusion, our study showed that non-ribosomally synthesized secondary metabolites of FZB42 are actually produced in the lettuce rhizosphere and contribute to the disease suppression by mediating plant defence gene expression towards the pathogen R. solani.