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Silver Nanoparticles (AgNps) have been widely used in the field of Medicine. This In Vitro study aimed to test (1) the bacterial viability after treating with AgNps and Chlorhexidine (CHX) against E. faecalis biofilm and (2) the anti-bacterial mechanism of action of AgNps. Sixty single rooted mandibular premolars were selected. The cylindrical midroot sections were enlarged with Gates Gliden dril no: 3. Dentin blocks were contaminated with E. faecalis (ATCC 29212). The samples were divided into three groups based on the medicament that was packed. Group 1: Saline (Control), Group II—2% CHX and Group III-AgNps. At the end of Days 1 and 3, assessment of live and dead cells were carried out by using confocal laser scanning microscopy (CLSM). The mechanism of antibacterial action was studied by membrane damage of E. faecalis, using Scanning Electron microscopy. It was also confirmed by membrane permeabilization assay. AgNps showed higher percentage of dead cells compared to CHX. There was no statistically significant difference between placing AgNps as a medicament for Day 1 and Day 3. The membrane damage of E. faecalis was proved after treating with AgNps.The current study proved the antibacterial efficacy of AgNps in the reduction of the E. faecalis biofilm.