Bacillus subtilis, a plant growth promoting rhizobacteria (PGPR), induces growth response and protection against pathogenic organisms through colonization and biofilm formation on the Arabidopsis thaliana root surface. In the current investigation, we utilized various Arabidopsis defense pathway mutants in a series of studies and showed that the plants recognize B. subtilis by a chemical-dependent cascade, which is independent of the salicylic acid (SA), jasmonic acid (JA), or ethylene pathways. These experiments revealed the importance of root surface chemistry in colonization and biofilm formation by B. subtilis. It was found that B. subtilis FB17 could not form biofilms on the roots of NahG, a transgenic Arabidopsis line for salicylate hydroxylase that produces catechol as the degradation product of SA. These findings suggest that catechol may play a direct role in inhibiting B. subtilis FB17 biofilm formation on the NahG root surface, possibly through induction of reactive oxygen species (ROS) in the roots. Using both in vitro microtitre plate and in planta assays we confirmed that catechol inhibited biofilm formation, but not the planktonic growth, of B. subtilis. Inhibition of biofilm formation was shown to be the result of a physiological response by B. subtilis to the presence of catechol, which resulted in the down-regulation of transcription of the yqxM-sipW-tasA and epsA-O operons, both of which are required for biofilm formation by B. subtilis. These data indicate that the suppression of biofilm formation on NahG plants was strongly influenced by the root-derived catechol production through ROS-mediated down-regulation of B. subtilis biofilm genes.