YycFG, one of the two-component systems involved in the regulation of biofilm formation, has attracted increasing interest as a potential target of antibacterial and antibiofilm agents. YycG inhibitors for <i>Staphylococcus aureus</i> and <i>Staphylococcus epidermidis</i> have been developed, but <i>Enterococcus faecalis</i> remains underexplored. Herein, we selected and identified novel candidate molecules against <i>E. faecalis</i> targeting histidine kinase YycG using high-throughput virtual screening; six molecules (compound-16, -30, -42, -46, -59, and -62) with low cytotoxicity toward mammalian cells were verified as potential YycG inhibitors through an autophosphorylation test and binding kinetics. Compound-16 inhibited planktonic cells of <i>E. faecalis</i>, including the vancomycin- or linezolid-resistant strains. In contrast, compound-62 did not affect planktonic growth but significantly inhibited biofilm formation in static and dynamic conditions. Compound-62 combined with ampicillin could synergistically eradicate the biofilm-embedded viable bacteria. The study demonstrates that YycG inhibitors may be valuable approaches for the development of novel antimicrobial agents for difficult-to-treat bacterial infections.