Composite nanofibers of polyethylene oxide (PEO)/bacterial cellulose whisker nanoparticles (BCW NPs) and PEO/functionalized bacterial cellulose whisker nanoparticles (f-BCW NPs) were fabricated by an electrospinning process. BCW NPs were prepared using a blender in order to increase the filler density in composite nanofibers. Nitrogen functionalization of the f-BCW NPs was performed by plasma treatment using a microwave oven. The chemical bonding between the nitrogen functional groups of f-BCW NPs and the hydroxyl groups of the polymer chains in the PEO matrix was enhanced by the nitrogen functionalization. The average diameter of the PEO/f-BCW NP composite nanofibers was slightly larger than that of PEO nanofibers and PEO/BCW NP composite nanofibers at the same concentration. The melting temperature (T-m), crystallization temperature (T-c), and weight loss of PEO/f-BCW NP composite nanofibers decreased compared to those of the PEO nanofibers and PEO/BCW NP composite nanofibers. This was a result of the f-BCW NPs hindering the crystallization of the PEO nanofiber structures. The strong interfacial interactions between the f-BCW NP nanofillers and the polymer matrix increased the glass transition temperature (T-g) compared to PEO nanofibers and PEO/BCW NP composite nanofibers. These results demonstrated that N-2 plasma treatment of BCW NPs embedded in PEO nanofiber was a useful tool for improving their thermal characteristics for biological applications.