Bacterial cellulose or Nata is produced using pineapple waste as substrate for the fermentation process by Acetobacter xylinum. This study was aimed to optimize the production of bacterial cellulose in static and agitated condition. Production of bacterial cellulose in agitated condition has been performed at different rpm, with and without adding micro particles. The fermentation conditions were set at pH 5.0 and 28 C. The highest bacterial cellulose formation is obtained from agitated condition with additional micro particles at 120 rpm whereas the bacterial cellulose weight is increased by 70.23 % compared to the static condition. The addition of microparticles helped in increasing the bacterial cellulose formation by 15.19 %. From the optimization using Design Expert(, yeast extracts and potassium dihydrogen phosphate (KH2PO4) are the most significant factors affecting the bacterial cellulose production. From the results, the optimum formulation for bacterial cellulose production from pineapple waste as medium were yeast extract 6 g, sucrose 20 g, bactopepton 1.49 g and KH2PO4 1.08 g for 176.47 g (wet weight) of bacterial cellulose production.