MgO-bacterial cellulose (BC) nanohybrids were fabricated by in-situ synthesis of nanoparticles (NPs) within BC network via two methods (the sonochemical and wet chemical). The ex-situ synthesized nanohybrid was prepared by immersing BC pellicles in the commercial MgO dispersion. The occurrence of new interactions between MgO-NPs and nanofibers was approved by Fourier transform infrared spectroscopy (FT-IR) spectra. X-ray diffraction (XRD) results indicated that the crystallinity index of nanofibers decreased after the formation of nanohybrid by the sonochemical in-situ method. The results of the field emission scanning electron microscopy (FE-SEM) indicated the formation of the small-sized NPs attached to the inner space of BC network at the in-situ synthesized nanohybrids. However, the agglomerated NPs precipitated on the surface of BC layer were observed for the ex-situ synthesized sample. The loading capacity of the ex-situ method was higher than that of the in-situ methods; but after 24 h, MgO releasing for in-situ and ex-situ synthesized nanohybrids was recorded about 16% and 28%, respectively. The antibacterial activity of the ex-situ synthesized nanohybrid against S. aureus and E. coli bacteria was more than those of both in-situ synthesized samples.