Bromelain is a protease, commercially derived from pineapple fruit or stem. In this study, bromelain was isolated from pineapple core (Ananas comosus [L.] Merr) then purified by fractionation using ammonium sulfate and acetone. The fractionated bromelain antibacterial activity was assayed against Staphylococcus epidermidis, Propionibacterium acnes, Staphylococcus aureus and Escherichia coli by disc diffusion method. The highest specific activity and purity was observed at the Fraction 2 ammonium sulfate with saturation level of 20-50%, and Fraction 3 acetone with 50-80% saturation level. The specific activity of Fraction 2 ammonium sulfate is 70 U/mg with a purity level of 5.3 fold compared to its crude extract. Meanwhile, the specific activity of the Fraction 3 acetone is 19.736 U/mg with a purity level of 2.5 fold compared to its crude extract. The antibacterial potency test showed that crude extract of bromelain has an antibacterial activity for all the tested bacteria. The antibacterial potency of the fractionated bromelain is only seen in P. acnes and S. aureus. Based on the antibacterial potency test results, fractionation using acetone can produce bromelain fraction with a better antibacterial activity compared to the fractionation using ammonium sulfate.