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<i>Enterococcus faecalis</i> is a common cause of biofilm-associated opportunistic infections, which are often difficult to treat. The formation of <i>E. faecalis</i> biofilms on the dentinal walls of the root canal is frequently the cause of endodontic treatment failure and secondary apical periodontitis. In a preliminary work, two recognized antifungal peptides, KP and L18R, showed antibacterial activity against planktonic <i>E. faecalis</i> cells at micromolar concentrations. Moreover, L18R proved to reduce the biomass in the early stage of <i>E. faecalis</i> biofilm development on polystyrene plates, while a qualitative biofilm inhibition was demonstrated on hydroxyapatite disks by confocal laser scanning microscopy (CLSM). The aim of this study was to better characterize the effect of both peptides on <i>E. faecalis</i> biofilm. A reduction in metabolic activity after peptide treatment was detected by Alamar Blue assay, while a remarkable impairment in the architecture of <i>E. faecalis</i> biofilms on hydroxyapatite disks, along with a significant reduction in viable bacteria, was caused mostly by L18R, as assessed by CLSM and scanning electron microscopy. The lack of cytotoxicity of the investigated peptides against L929 murine fibroblasts was also determined. Obtained results suggest L18R as a promising candidate for the development of new strategies for endodontic infection control.