Bacterial cellulose (BC) has garnered attention among biomaterial scientists for its unique physicochemical features and biocompatibility; however, the lack of bioactivity has limited its biomedical applications. Thus, this study describes the in situ preparation of BC/hydroxyapatite (HA) nanocomposite membranes using static and agitated fermentation to enhance the bioactivity of BC. The incorporation of HA increased BC production from 2.31 g/L without HA to 4.10 and 3.26 g/L under static and agitated fermentation, respectively, although the SEM observation indicates the formation of a fibrous structure in BC mesh under both fermentations. It was also observed that the content of HA nanoparticles in BC obtained from agitated fermentation was higher than that obtained from static fermentation. In addition, the average fiber diameter was increased from 56 ± 17 nm for agitated nanocomposites (BC/HA-A) to 145 ± 48 nm for static BC/HA nanocomposites (BC/HA-S) and 122 ± 26 nm for BC. In conclusion, the in situ formation of BC/HA nanocomposite under agitated fermentation appears more convenient in term of BC yield, HA content and distribution, and cytotoxicity against fibroblast cells (BJ1). This strategy will inspire new ways to prepare BC-based materials for medical applications.