B Background: Bromelain is a sulfhydryl proteolytic enzyme that can hydrolyze protein, protease or peptide. Bromelain is composed of 212 amino acid residues with cysteine-25 fom1ing a polypeptide chain that can hydrolyze using 1-120. In medicine, bromelain has been developed as antibiotic, cancer drug, anti-inflammatory and immunomodulator. Ln dentistry, bromelain has potential to reduce plaque formation and to irigate root canal. Objective: to know the characteristics of bromelain isolate Ananas comosus and evaluate the effect on Enterococcus faecalis. Methods: Pineapple core was dried and extracted with water for 24 hours using maceration method. After that, do purification using ammonium sulfate fractionation, dialysis, ion exchange chromatography, Sodium Dodecyl Sulphate - polyacrylamide gel electrophoresis (SOS-PAGE) and zimogram. To determine the effect on Enlerococcus faeca/is using diffusion method. Resulb: The specific activity of bromelain after ammonium sulfate fractionation, dialysis and ion exchange chromatography was 44.79 U/mg, 81.96 U/mg and 344.64 U/mg. Fold of bromelain after ammonium sulfate fractionation, dialysis and ion exchange chromatography was 9.40, 17.21 and 72.40. The identification showed by SOS-PAGE has molecular weight on 33 kDa and zimogram showed the existence of a clear zone because degradation of proteolytic enzyme on 19.2 kDa and 34.1 kDa. The results of 2 ways ANOV A between concentration and time groups there is significant difference (F:566.41, p<0.000) and (F:8.27, p<0.001) while there was no interaction between concentration and time group (F:0.81, p<0.596). Conclusion: Bromclain isolate Ananas comosus has specific activity higher than crude extract and ability to inhibit En1erococcusfaecalis at concentration of 12.5%.