Background- Persistent infection of the root canal due to the presence of resistance bacterial species, such as Entero-coccus faecalis, has always been one of the most important reasons for endodontic treatment failure. Aim- To evaluate the bactericidal effects of linezolid, lantibiotic (nisin), and calcium hydroxide against Enterococcus faecalis biofilm. Methods- Single rooted human mandibular premolars were decoronated, biomechanically prepared, and vertically sectioned along the midsagittal plane to obtain a standardized tooth substrate. Standardized suspension of E. faecalis and tooth substrate was incubated for 3 weeks to allow growth of biofilm. At the end of 3 weeks, the grouping was done according to the medicament used, Group A-Linezolid, Group B-Lantibiotic (nisin), Group C-Calcium hydroxide, Group D-Negative treatment. The zones of inhibition were checked after 2 and 7 days. Results- Group A gave the widest zone of inhibition (17±1.14 and 17±1.06) followed by Group B (12±1.68 and 12±2.03) and Group C (3.78±0.64 and 2.56±0.48) both after 2 and 7 days. The antimicrobial efficacy against E. faecalis biofilm remain unchanged for Group A and B after 7 days whereas in Group C, the zone of inhibition decreased after 7 days. Conclusion- Linezolid showed maximum antimicrobial potential against E. faecalis biofilm followed by lantibiotic (nisin) after 2 and 7 days. Antimicrobial efficacy against E. faecalis biofilm reduced with lapse of time for calcium hydroxide.