OBJECTIVE To evaluate, in vitro, the antimicrobial activity and biofilm formation of three chlorhexidine varnishes in four E. faecalis strains: E. faecalis ATCC 29212, E. faecalis EF-D1 (from failed endodontic treatment), E. faecalis 072 (cheese) and E. faecalis U-1765 (nosocomial infection), and one E. durans strain (failed endodontic treatment). METHODS The direct contact test was used to study the antimicrobial activity. Bacterial suspensions were exposed for one hour to EC40, Cervitec (CE) and Cervitec Plus (CEP) varnishes. "Eradication " was defined as 100% bacterial kill. The formation of enterococci biofilms was tested on the surface of the varnishes after 24 hours of incubation and expressed as percentage of biofilm reduction. RESULTS EC40 eradicated all strains except E. faecalis ATCC 29212, where 98.78% kill was achieved. CE and CEP showed antimicrobial activity against all the strains, but most clearly against E. durans and E. faecalis 072. EC40 completely inhibited the formation of biofilm of E. faecalis ATCC 29212, E. faecalis 072 and E. durans. CE and CEP led to over 92% of biofilm reduction, except in the case of E. faecalis U-1765 on CEP (76.42%). CONCLUSIONS The three varnishes studied were seen to be effective in killing the tested strains of enterococci and in inhibiting the formation of biofilm, the best results being observed with EC40.