This study was conducted to isolate and purify bromelain from pineapple core (Ananas comosus (L.) Merr.) by multisteps fractionation using acetone and ammonium sulphate. The antibacterial activity of bromelain fraction with the highest specific activity was examined using disc diffusion method against S. mutans and P. gingivalis, bacteria that cause dental caries and periodontal disease. The crude enzyme showed proteolitic activity with the specific activity of 52.318 Unit/mg. Fractionation of crude enzyme using acetone produced the third fraction with level of saturation of 50-80 % that has a highest specific activity of 87.778 Unit/mg. Further purification by fractionation using ammonium sulphate produced the third fraction which has a specific activity value increase to 260 Unit/mg. After dialysis, the specific activity of the ammonium sulphate third fraction was increased to 340.926 Unit/mg. The antibacterial assay showed that ammonium sulphate fraction has a strong activity against P. gingivalis bacteria with diameter of clear zone of 21 mm and weak activity against S. mutans with diameter of clear zone of 12 mm.