Enterococcus faecalis is a gram-positive, rod-shape bacteria responsible for around 65% to 80% of all enterococcal nosocomial infections. It is multidrug resistant (MDR) bacterium resistant to most of the first-line antibiotics. Due to the emergence of MDR strains, there is an urgent need to find novel targets to develop new antibacterial drugs against E. faecalis. In this regard, we have identified naphthoate synthase (1,4-dihydroxy-2-naphthoyl-CoA synthase, EC: 4.1.3.36; DHNS) as an anti-E. faecalis target, as it is an essential enzyme for menaquinone (vitamin K ) synthetic pathway in the bacterium. Thus, inhibiting naphtholate synthase may consequently inhibit the bacteria's growth. In this regard, we report here cloning, expression, purification, and preliminary structural studies of naphthoate synthase along with in silico modeling, molecular dynamic simulation of the model and docking studies of naphthoate synthase with quercetin, a plant alkaloid. Biochemical studies have indicated quercetin, a plant flavonoid as the potential lead compound to inhibit catalytic activity of EfDHNS. Quercetin binding has also been validated by spectrofluorimetric studies in order to confirm the bindings of the ligand compound with EfDHNS at ultralow concentrations. Reported studies may provide a base for structure-based drug development of antimicrobial compounds against E. faecalis.