Bacilysin, formed by L-alanine and L-anticapsin, is also the simplest structured peptide antibiotic known to be synthesized nonribosomally by some strains of Bacillus subtilis. In our previous study, to elucidate the functional role of bacilysin in the producer organism, a genome-wide comparative transcriptome analysis was performed between the wild-type strain PY79, as a natural bacilysin producer, and its bacilysin non-producer derivative strain. The results of this global analysis strongly suggested that bacilysin can be a pleiotropic regulatory factor that affected the expression of various physiological and adaptive cellular processes such as competence development, sporulation, and biofilm formation. As further work, in this project, we aimed to elucidate the direct regulatory role of bacilysin. For this, comparative transcriptome analysis was performed in the presence and absence of bacilysin by next-generation sequencing (RNA-seq) to elucidate the differentially expressed genes in the presence of bacilysin. Subsequently, by performing bioinformatics analysis, the functional role and metabolic activities of the biofilm-related genes were identified, and thereby, the possible regulatory role of bacilysin during the biofilm formation process was elucidated.