– Gramicidin S is an anttiboitics. Gramicidin S reduces the cell number of planktonic cells within 20-40 min at a concentration of 40-80 ?g/mL. Gramicidin S kills the cells of pre-grown biofilms at concentrations of 100-200 ?g/mL, such that no re-growth is possible. The translocation of the peptide into the cell interior and its complexation with intracellular nucleotides, including the alarmon ppGpp, can explain its anti-biofilm effect. The successful treatment of persistently infected root canals of two volunteers confirms the high effectiveness of GS. The broad Gramicidin S activity towards resistant, biofilm-forming E. faecalis suggests its applications for approval in root canal medication.