This in vitro study developed and evaluated a novel intracanal medication by loading calcium hydroxide onto a Gum Arabic nanoparticle (GA NP) carrier to improve its antibacterial efficacy against Enterococcus faecalis biofilms. Nanoparticles were characterized using TEM and FTIR, and their antibacterial performance was assessed by colony-forming unit (CFU) counting, quantitative PCR, scanning electron microscopy (SEM), and analysis of biofilm-associated genes (agg, ace, and efaA). The calcium hydroxide-loaded Gum Arabic nanoparticles significantly reduced bacterial counts, downregulated biofilm-related gene expression, and demonstrated superior antibiofilm activity compared with conventional calcium hydroxide and other tested formulations. These findings suggest that this nanoparticle-based formulation is a promising intracanal medicament for managing persistent endodontic infections caused by E. faecalis.