ABSTRACT In this study, we have optimized production of bacterial cellulose (BC) by Komagataeibacter hansenii ATCC 23769 in a static cultivation using sisal juice, an agroindustrial residue, as substrate. Optimization of fermentation parameters has been carried out using the one-variable-at-a-time method. Effect of initial sugar concentration, pH, nitrogen supplement, and cultivation time was evaluated. The influence of nitrogen source and quantity for bacterial cellulose production was studied using a central composite rotational design (CCRD).The highest production of BC (3.38 g/L) was obtained after 10 days of cultivation, using sisal juice (pH 5) at 15 g/L of sugars and supplemented with 7.5 g/L of extract yeast. The cellulose production yield in selected sisal culture conditions was three times higher than the yield in synthetic medium, indicating that sisal juice is a suitable substrate for BC production.