Gramicidin S 2HCl, is an antibiotic. At concentrations of 40-80 μg/mL, GS can reduce planktonic cell numbers within 20-40 minutes. For established biofilms, it effectively kills cells at concentrations of 100-200 μg/mL without regrowth. This anti-biofilm effect results from peptide transmembrane transport and complexation with intracellular nucleotides (including the alarmone ppGpp). GS demonstrates wide-ranging activity against biofilm-forming, drug-resistant infections caused by E. faecalis, proving its efficacy in endodontic treatment. The successful treatment of persistent infections in two volunteer cases further confirms GS's potent therapeutic performance. These studies indicate that GS can be applied in certifying compounds used in endodontics.