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 Staphylococcus aureusand Staphylococcus epidermidishave been developed, but Enterococcus faecalisremains underexplored. Herein, we selected and identified novel candidate molecules against E. faecalistargeting 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 E. faecalis, 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.