The demand for safe and effective endodontic medicaments to control Enterococcus faecalis biofilms, a contributor to apical periodontitis, is increasing. Recently, lipoteichoic acid (LTA) of family Lactobacillaceae has been shown to have anti-biofilm effects against various oral pathogens. Preliminary experiments showed that LTA purified from Lacticaseibacillus rhamnosus GG (Lgg.LTA) was the most effective against E. faecalis biofilms among LTAs from three Lactobacillaceae including L. rhamnosus GG, Lacticaseibacillus casei, and Lactobacillus acidophilus. Therefore, in this study, we investigated the potential of Lgg.LTA as an intracanal medicament in human root canals infected with E. faecalis. Twenty eight dentinal cylinders were prepared from extracted human teeth, where two-week-old E. faecalis biofilms were formed followed by intracanal treatment with sterile distilled water (SDW), N-2 methyl pyrrolidone (NMP), calcium hydroxide (CH), or Lgg.LTA. Bacteria and biofilms that formed in the root canals were analyzed by scanning electron microscopy and confocal laser scanning microscopy. The remaining E. faecalis cells in the root canals after intracanal medicament treatment were enumerated by culturing and counting. When applied to intracanal biofilms, Lgg.LTA effectively inhibited E. faecalis biofilm formation as much as CH, while SDW and NMP had little effect. Furthermore, Lgg.LTA reduced both live and dead bacteria within the dentinal tubules, indicating the possibility of minimal re-infection in the root canals. Collectively, intracanal application of Lgg.LTA effectively inhibited E. faecalis biofilm formation, implying that Lgg.LTA can be used as a novel endodontic medicament.