The aim of this study was to investigate the antibacterial activity against Enterococcus faecalis of alpha-mangostin and to compare its activity with sodium hypochlorite (NaOCl) and chlorhexidine (CHX) as well as to assess its biocompatibility using human periodontal ligament (PDL) cell.Alpha-mangostin was extracted from the pericarps of mangosteen by thin layer chromatography. The inhibitory effect of alpha-mangostin on E. faecalis was determined by cultures and an applied broth dilution test. The rate of bactericidal activity was evaluated by time-kill. Human PDL cell culture was used to assess the cytotoxicity of alpha-mangostin.Alpha-mangostin was effective against E. faecalis, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were 1.97 and 3.94 μg/ml, respectively. The E. faecalis killing activity of alpha-mangostin was higher than that of CHX at 2 × MBC and 4 × MBC, respectively. When compared at the same concentration, there was no significant difference between the alpha-mangostin-treated group and the NaOCl-treated group (P < 0.05). From the cytotoxicity test demonstrated that no significant difference of cell viability was observed between the alpha-mangostin-treated group and control group in all of the treatment time intervals.These findings indicated that alpha-mangostin showed promising antibacterial activity against E. faecalis and low toxicity to human PDL cells in vitro, suggesting its potential to be used as a root canal irrigant.