Root canal disinfection is one of the most important factors governing success of root canal treatment, especially when regenerative strategies are used. This study evaluated the efficacy of 5 intracanal medicaments against mature biofilms of Enterococcus fecalis in vitro: Light activated curcumin, triple antibiotic paste (TAP), double antibiotic paste (DAP), chlorhexidine, calcium hydroxide. Untreated teeth with biofilms served as controls. Confocal microscopy was used to analyse the biofilm mass and percentage of live/dead bacteria within the root canal as well as dentinal tubules. Dentinal shavings obtained from the root canal walls (at 200 and 400 microns depth) were used to quantify the colony forming units/mL. The results showed that light activated curcumin and triple antibiotic paste brought about complete disruption of the biofilm structure (P < 0.05) while chlorhexidine and calcium hydroxide were not significantly different from the control (P > 0.05). Light activated curcumin brought about the highest percentage of dead cells at both depths, but this was not significantly different from triple antibiotic paste (P > 0.05). Curcumin, TAP and DAP brought about a significant reduction of CFU/mL at both depths compared to the control and other groups (P < 0.05). Light activated curcumin brought about a 7 log reduction of bacteria at both depths.