Bacitracin has well familiar effects on growth and colonization of bacteria but its antibiofilm action on majority of bacteria is still not studied. Bacitracin is a bactericidal antibiotic that primarily acts on Gram positive bacteria by obstructing the process of cell wall synthesis. In this study, we have investigated antibiofilm potential and the mechanism of bacitracin against a cariogenic bacteria ‘Streptococcus mutans’ which has not been reported so far. Bacitracin has been found to affect propensity of S. mutans to form biofilm. On treatment with sub-MIC concentration of bacitracin resulted in significant reduction in bifilm formation as evaluated by crystal violet and congo red assays. The architecture of S. mutans biofilm was observed by scanning electron microscopy which revealed astonishing phenotype of biofilm. Deficient biofilm was found to be composed of abnormally elongated cells. Transmission electron microscopy showed multiple septa formation in each cell of biofilm thereby indicating, cell division defect as the most probable cause of cell elongation. To elucidate the effect of bacitracin on molecular level, expression profiling of genes critically important for cell division and biofilm formation was performed, which were found many folds downregulated. Bacitracin at very low concentration has been found to have potent antibiofilm activity, therefore is a potential antibiofilm agent to treat oral biofilms. It is being anticipated, this study will offer novel information to identify potential targets and effectively creates true innovation to understand the biofilm's basic biology. Besides, discovering new uses for currently marketed drugs makes commercial as well as research sense.