AIM This study investigated the antimicrobial activity of taurolidine and N-acetylcysteine (NAC) on planktonic and biofilm Enterococcus faecalis phenotypes. MATERIALS AND METHODS The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of NAC and taurolidine were determined using broth microdilution, utilizing calcium hydroxide (CH), sodium hypochlorite, and chlorhexidine for comparisons. Thereafter, the ability of dentin powder to neutralize the antibacterial activity of NAC and taurolidine was studied. The efficacy of both antimicrobial agents on E. faecalis biofilms was examined quantitatively by exposure of 21-day-old E. faecalis biofilms on dentin disks. The cytotoxicity of human dental pulp fibroblast cells in contact with the extracts was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS The MIC and MBC of NAC, taurolidine, and CH were not affected by pre-incubation in dentin powder. As verified by qualitative assay of E. faecalis biofilms, CH was the strongest bactericidal agent at all test dilutions, regardless of the presence of dentin powder. The antibacterial effect of NAC and taurolidine was significantly lower than that of CH at all test dilutions. At 48 h, all test agents showed similar, but high levels of cytotoxicity. CONCLUSION NAC and taurolidine were effective against E. faecalis in planktonic state, at the expense of demonstrating cytotoxic effects. For both planktonic and biofilm forms of E. faecalis, neither NAC nor taurolidine offered any advantage over CH.