Due to its properties, such as high superficial area, high porosity, tensile strength and elevated adsorption capacity, the bacterial cellulose (BC) can be applied in different fields. The present study aimed to evaluate, through a central composite rotatable design (CCRD), the effects of the agitation rate and inoculum dose on the diameter of BC spheres from Komagataeibacter hansenii, cultivated in environments containing mannitol as a carbon source at 30 ºC of temperature. The only statistically significant factor (p < 0.05) was found to be the agitation rate for a 95% confidence level, with a negative linear effect. CCRD study showed that the sphere diameter decreases when the agitation rate increases at any inoculum dosage. However, to a given agitation, the sphere size initially decreases and then increases with the increase of the inoculum dose. Increases in the agitation rate also led to an increase in the sphere number at a constant inoculum dosage. The results contribute to explain the controverted results found in the literature about the influence of these two parameters. From the optimization of the processing parameters via CCRD, the selection of the most adequate parameter values to obtain spherical BC of the desired diameter can be achieved.